Modified release pharmaceutical formulation

ABSTRACT

A modified release pharmaceutical composition comprising, as active ingredient, a compound of formula (I), wherein R 1  represents C?1-2#191 alkyl substituted by one or more fluoro substituents; R 2  represents hydrogen, hydroxy, methoxy or ethoxy; and n represents 0, 1 or 2; or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable diluent or carrier, provided that the formulation may only contain iota-carrageenan and a neutral gelling polymer when the compound of formula (I) is in the form of a salt; such formulations being of use for the treatment of a cardiovascular disorder

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 of PCTapplication PCT/SE03/00858 filed May 27, 2002, which claims priorityfrom Sweden Application No. 0201659-0, filed May 31, 2002, thespecifications of each of which are incorporated by reference herein.

This invention relates to novel modified release pharmaceuticalformulations that provide for modified delivery of particularpharmaceuticals, to the manufacture of such formulations, and to the useof such a formulation in the treatment or prevention of thrombosis.

It is often necessary to administer pharmaceutically active compoundsfrequently throughout the day in order to maintain a desired therapeuticlevel of active principle in plasma, body tissues and/or thegastrointestinal tract. This is particularly the case where it isintended to deliver the drug orally and to provide a uniform responseover an extended period of time.

Over the last thirty or so years, modified release dosage forms haveincreasingly become a preferred method of delivering certain drugs topatients, particularly via the oral route. Such forms may for exampleprovide for release of drug over an extended period of time, thusreducing the number of required daily doses, and during which time therate of release may be substantially uniform and/or constant, within aspecific part of the gastrointestinal tract, or pulsative.

There are numerous modified release dosage forms known in the art andthese have been summarised by inter alia De Haan and Lerk inPharmaceutisch Weekblad Scientific Edition, 6, 57 (1984); Banker in“Medical Applications of Controlled Release”, Vol II, eds. Langer andWise (1984) Bocaraton, Fla., at pages 1 to 34; Graffner in IndustrialAspects of Pharmaceuticals, ed. Sandel, Swedish Pharmaceutical Press(1993) at pages 93 to 104; and Proudfoot “Dosage Regimens: TheirInfluence on the Concentration-Time Profile of the Drug in the Body” atpages 191 to 211 of “Pharmaceutics: The Science of Dosage Form Design”,ed. M. E. Aulton (1988) (Churchill Livingstone).

International Patent Application No. PCT/SE01/02657 (WO 02/44145,earliest priority date 1 Dec. 2000, filed 30 Nov. 2001, published 6 Jun.2002) discloses a number of compounds that are, or are metabolised tocompounds which are, competitive inhibitors of trypsin-like proteases,such as thrombin. The following three compounds are amongst those thatare specifically disclosed:

-   (a) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe):

which compound is referred to hereinafter as Compound A;

-   (b) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe):

which compound is referred to hereinafter as Compound B; and

-   (c) Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe):

which compound is referred to hereinafter as Compound C.

The methoxyamidine Compounds A, B and C are metabolised following oraland/or parenteral administration to a mammal and form the correspondingfree amidine compounds, which latter compounds have been found to bepotent inhibitors of thrombin. Thus:

-   Compound A is metabolized to    Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab (which compound is    referred to hereinafter as Compound D) via a prodrug intermediate    Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OH) (which compound is    referred to hereinafter as Compound G);-   Compound B is metabolized to    Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF) (which compound    is referred to hereinafter as Compound E) via a prodrug intermediate    Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH) (which    compound is referred to hereinafter as Compound H); and,-   Compound C is metabolized to    Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab (which compound is    referred to hereinafter as Compound F) via a prodrug intermediate    Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OH) (which compound is    referred to hereinafter as Compound J).

Processes for the synthesis of Compounds A, B, C, D, E, F, G and J aredescribed in Examples 12, 40, 22, 3, 39, 21, 2 and 31 (respectively) ofinternational patent application No. PCT/SE01/02657. A modified releaseformulation of these compounds, or their metabolites has yet to bedescribed in the literature.

We have already found that Compounds A and C can be formulated incertain modified release iota-carrageenan formulations, and have nowfound that the compounds of formula (I) and their salts can beformulated in other modified release pharmaceutical formulations whichare easy to administer, for example by oral administration.

According to a first aspect of the invention, there is provided amodified release pharmaceutical formulation comprising, as activeingredient, a compound of formula (I):

-   R¹ represents C₁₋₁₂ alkyl substituted by one or more fluoro    substituents;-   R² represents hydrogen, hydroxy, methoxy or ethoxy; and-   n represents 0, 1 or 2;    or a pharmaceutically acceptable salt thereof; and a    pharmaceutically acceptable diluent or carrier; provided that the    formulation may only contain iota-carrageenan and a neutral gelling    polymer when the compound of formula (I) is in the form of a salt;    which formulations are referred to hereinafter as “the formulations    of the invention”.

The compounds of formula (I), or a pharmaceutically acceptable saltthereof, may be in the form of a solvate, a hydrate, a mixedsolvate/hydrate or, preferably, an ansolvate, such as an anhydrate.Solvates may be of one or more organic solvents, such as lower (forexample C₁₋₄) alkyl alcohols (for example methanol, ethanol oriso-propanol), ketones (such as acetone), esters (such as ethyl acetate)or mixtures thereof.

In one particular aspect of the invention R¹ is CHF₂ or CH₂CH₂F.

The variable n is preferably 0 or 2.

More preferred compounds of formula (I) include those in which nrepresents 0, or those in which n represents 2, so providing two fluoroatoms located at the 2- and 6-positions (that is the two ortho-positionsrelative to the point of attachment of the benzene ring to the —NH—CH₂—group).

The compound of formula (I) is especially Compound A, Compound B orCompound C.

A neutral gelling polymer is a single, or a mixture of more than one,neutral erodable polymer(s) having gelling properties and havingsubstantially pH-independent solubility.

Preferred salts of the compounds of formula (I) are acid addition salts.Acid addition salts include inorganic acid addition salts, such as thoseof sulphuric acid, nitric acid, phosphoric acid and hydrohalic acids,such as hydrobromic acid and hydrochloric acid. More preferred acidaddition salts include those of organic acids, such as those ofdimethylphosphoric acid; saccharinic acid; cyclohexylsulfamic acid;those of carboxylic acids (such as maleic acid, fumaric acid, asparticacid, succinic acid, malonic acid, acetic acid, benzoic acid,terephthalic acid, hippuric acid, 1-hydroxy-2-naphthoic acid, pamoicacid, hydroxybenzoic acid and the like); those of hydroxy acids (such assalicylic acid, tartaric acid, citric acid, malic acid (includingL-(−)-malic acid and, D,L-malic acid), gluconic acid (includingD-gluconic acid), glycolic acid, ascorbic acid, lactic acid and thelike); those of amino acids (such as glutamic acid (includingD-glutamic, L-glutamic, and D,L-glutamic, acids), arginine (includingL-arginine), lysine (including L-lysine and L-lysine hydrochloride),glycine and the like); and, particularly, those of sulfonic acids, (suchas 1,2-ethanedisulfonic acid, camphorsulfonic acids (including1S-(+)-10-camphorsulfonic acid and (+/−)-camphorsulfonic acids),ethanesulfonic acid, a propanesulfonic acid (including n-propanesulfonicacid), a butanesulfonic acid, a pentanesulfonic acid, a toluenesulfonicacid, methanesulfonic acid, p-xylenesulfonic acid, 2-mesitylenesulfonicacid, naphthalenesulfonic acids (including 1,5-naphthalenesulfonic acidand naphthalenesulfonic acid), benzenesulfonic acid,hydroxybenzenesulfonic acids, 2-hydroxyethanesulfonic acid,3-hydroxyethanesulfonic acid and the like).

Particularly preferred salts include those of C₁₋₆ (for example C₁₋₄)alkanesulfonic acids, such as ethanesulfonic acid (esylate) andpropanesulfonic acid (for example n-propanesulfonic acid) and optionallysubstituted (for example with one or more C₁₋₂ alkyl groups)arylsulfonic acids, such as benzenesulfonic acid (besylate) andnaphthalenedisulfonic acid.

Suitable stoichiometric ratios of acid to free base are in the range0.25:1.5 to 3.0: 1, such as 0.45:1.25 to 1.25:1, including 0.50:1 to1:1.

According to a further aspect of the invention there is providedformulation comprising a compound of formula (I) in substantiallycrystalline form.

Although we have found that it is possible to produce compounds of theinvention in forms which are greater than 80% crystalline, by“substantially crystalline” we include greater than 20%, preferablygreater than 30%, and more preferably greater than 40% (e.g. greaterthan any of 50, 60, 70, 80 or 90%) crystalline.

According to a further aspect of the invention there is also provided acompound of the invention in partially crystalline form. By “partiallycrystalline” we include 5% or between 5% and 20% crystalline.

The degree (%) of crystallinity may be determined by the skilled personusing X-ray powder diffraction (XRPD). Other techniques, such as solidstate NMR, FT-IR, Raman spectroscopy, differential scanning calorimetry(DSC) and microcalorimetry, may also be used.

Preferred compounds of formula (I) that may be prepared in crystallineform include salts of C₁₋₆ (for example C₂₋₆, such as C₂₋₄)alkanesulfonic acids, such as ethanesulfonic acid, propanesulfonic acid(for example n-propanesufonic acid) and optionally substitutedarylsulfonic acids, such as benzenesulfonic acid andnaphthalenedisulfonic acid.

The term “modified release” pharmaceutical composition will be wellunderstood by the skilled person to include any composition/formulationin which the onset and/or rate of release of drug is altered by galenicmanipulations, and thus includes the definition provided in the UnitedStates Pharmacopeia (USP XXII) at pages xliii and xliv of thepreface/preamble part, the relevant disclosure in which document ishereby incorporated by reference.

In the present case, modified release may be provided for by way of anappropriate pharmaceutically-acceptable carrier, and/or other means,which carrier or means (as appropriate) gives rise to an alteration ofthe onset and/or rate of release of active ingredient. Thus, the termwill be understood by those skilled in the art to include compositionswhich are adapted (for example as described herein) to provide for a“sustained”, a “prolonged” or an “extended” release of drug (in whichdrug is released at a sufficiently retarded rate to produce atherapeutic response over a required period of time, optionallyincluding provision for an initial amount of drug being made availablewithin a predetermined time following administration to cause an initialdesired therapeutic response); compositions which provide for a“delayed” release of drug (in which the release of drug is delayed untila specific region of the gastrointestinal tract is reached, followingwhich drug release may be either pulsatile or further modified asindicated above); as well as so-called “repeat action” compositions (inwhich one dose of drug is released either immediately or some time afteradministration and further doses are released at a later time).

We prefer that the compositions of the invention provide for a delayedrelease or, more preferably, a sustained (that is prolonged or extended)release of drug over a period of time. More preferred compositions ofthe invention may be adapted (for example as described herein) toprovide a sufficient dose of drug over the dosing interval (irrespectiveof the number of doses per unit time) to produce a desired therapeuticeffect. Release may be uniform and/or constant over an extended periodof time, or otherwise.

Compositions of the invention may, for example, be in the form of thefollowing, all of which are well known to those skilled in the art:

-   (a) Coated pellets, tablets or capsules, which may be designed to    release at least some of the drug when the formulation in question    reaches a particular region of the gastrointestinal tract. Such    tablets may, for example be provided with some form of    gastro-resistant coating, such as an enteric coating layer,    providing for release of at least part of the drug present in the    formulation in a specific part of the gastrointestinal tract, such    as the intestinal regions.-   (b) Multiple unit or multiparticulate systems, which may be in the    form of microparticles, microspheres or pellets comprising drug    (which multiple units/multiparticulates may provide for gradual    emptying of the formulation containing drug from the stomach into    the duodenum and further through the small and large intestine while    releasing drug at a pre-determined rate).-   (c) Formulations comprising dispersions or solid solutions of active    compound in a matrix, which may be in the form of a wax, gum or fat,    or, particularly, in the form of a polymer, in which drug release    takes place by way of gradual surface erosion of the tablet and/or    diffusion.-   (d) Systems which comprise a bioadhesive layer, which layer may    provide for prolonged retention of composition of the invention in a    particular region of the gastrointestinal tract (for example the    stomach). This includes floating or sinking systems (that is low and    high density systems, respectively), as well as so-called    “volume-enlarging” systems.-   (e) So-called “pendent” devices, in which drug is attached to an ion    exchange resin, which provides for gradual release of drug by way of    influence of other ions present in the gastrointestinal tract, for    example, the acid environment of the stomach.-   (f) Devices in which release rate of drug is controlled by way of    its chemical potential (for example the Osmotic Pump).-   (g) Systems in which drug is released by diffusion through    membranes, including multilayer systems.-   (h) Devices that act in accordance with an external signal, to    release a small amount of drug.-   (i) Active, self-programmed systems, which may contain a sensing    element, which element responds to a particular biological    environment to modulate drug delivery.-   (j) Silastic controlled release depots, which release drug as a    function of diffusion of water and/or gastrointestinal fluids into    the device via an entry/exit port, resulting in dissolution and    subsequent release of drug.

The above principles are discussed at length in prior art referencesincluding Pharmaceutisch Weekblad Scientific Edition, 6, 57 (1984);Medical Applications of Controlled Release, Vol II, eds. Langer and Wise(1984) Bocaraton, Fla., at pages 1 to 34; Industrial Aspects ofPharmaceuticals, ed. Sandel, Swedish Pharmaceutical Press (1993) atpages 93 to 104; and pages 191 to 211 of “Pharmaceutics: The Science ofDosage Form Design”, ed. M. E. Aulton (1988) (Churchill Livingstone); aswell as the references cited in the above-mentioned documents, thedisclosures in all of which documents are hereby incorporated byreference.

In another aspect the present invention provides an oral modifiedrelease formulation wherein R² is hydroxy or methoxy, (such as CompoundA, B, C, G, H or J; R² is especially methoxy, for example Compound A, Bor C) or a pharmaceutically acceptable salt thereof (especially acrystalline salt thereof; such as a C₁₋₆ (for example C₂₋₆, such asC₂₋₄) alkanesulfonic acid salt, or an optionally substitutedarylsulfonic acid salt).

The invention includes parenteral modified release formulations usingcompounds of formula (I). In a further aspect the present inventionprovides a parenteral modified release formulation wherein R² ishydrogen (such as Compound D, E or F).

In a still further aspect the invention provides a modified releaseformulation which comprises a gelling matrix. The matrix preferablycomprises hydroxy propyl methyl cellulose (HPMC), iota-carrageenan,sodium dodecyl sulphate (SDS) and/or xanthan gum. More preferably thematrix comprises hydroxy propyl methyl cellulose (HPMC),iota-carrageenan and/or PEO. The HPMC may be one or a mixture of two ormore HPMCs of different viscosities or molecular weights (as describedanywhere below).

The invention also provides a modified release formulation comprisingone or more HPMCs and one or more further components selected from thegroup comprising: iota-carrageenan, microcrystalline cellulose, alubricant (such as sodium stearyl fumarate) or mannitol.

The invention further provides, in a further aspect, a modified releaseformulation comprising xanthan gum; or comprising iota-carrageenan andPEO (as described below).

Suitable modified release formulations may thus be prepared inaccordance with standard techniques in pharmacy, as described herein orin the above-mentioned documents, and/or which are well known.

We prefer that, in the compositions of the invention, active ingredientis provided together with a pharmaceutically acceptable carrier. Inparticular, we prefer that compositions of the invention are presentedin the form of active ingredient in a polymer matrix.

In this respect, we prefer that the compositions of the invention areprovided for oral administration in the form of a so-called “swelling”modified-release system, or a “gelling matrix” modified-release system,in which active ingredient is provided together with a polymer thatswells in an aqueous medium (that is a “hydrophilic gelling component”).The term “aqueous medium” is to be understood in this context to includewater, and liquids which are, or which approximate to, those present inthe gastrointestinal tract of a mammal. Such polymer systems typicallycomprise hydrophilic macromolecular structures, which in a dry form maybe in a glassy, or at least partially crystalline, state, and whichswell when contacted with aqueous media. Modified release of drug isthus effected by one or more of the following processes: transport ofsolvent into the polymer matrix, swelling of the polymer, diffusion ofdrug through the swollen polymer and/or erosion of the polymer, one ormore of which may serve to release drug slowly from the polymer matrixinto an aqueous medium.

Thus, suitable polymeric materials (acting as carriers), which may beused as the hydrophilic gelling component of a gelling matrixmodified-release composition include those with a molecular weight ofabove 5000 g/mol, and which either:

-   (a) are at least sparingly soluble in; or-   (b) swell when placed in contact with,    aqueous media (as defined hereinbefore), so enabling release of drug    from the carrier.

Suitable gelling matrix polymers, which may be synthetic or natural,thus include polysaccharides, such as maltodextrin, xanthan,scleroglucan dextran, starch, alginates, pullulan, hyaloronic acid,chitin, chitosan and the like; other natural polymers, such as proteins(albumin, gelatin etc.), poly-L-lysine; sodium poly(acrylic acid);poly(hydroxyalkylmethacrylates) (for examplepoly(hydroxyethylmethacrylate)); carboxypolymethylene (for exampleCarbopol™); carbomer; polyvinylpyrrolidone; gums, such as guar gum, gumarabic, gum karaya, gum ghatti, locust bean gum, tamarind gum, gellangum, gum tragacanth, agar, pectin, gluten and the like; poly(vinylalcohol); ethylene vinyl alcohol; poly(ethylene oxide) (PEO); andcellulose ethers, such as hydroxymethylcellulose (HMC),hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC),methylcellulose (MC), ethylcellulose (EC), carboxyethylcellulose (CEC),ethylhydroxyethylcellulose (EHEC), carboxymethylhydroxyethylcellulose(CMHEC), hydroxypropylmethyl-cellulose (HPMC),hydroxypropylethylcellulose (HPEC) and sodium carboxymethylcellulose (NaCMC); as well as copolymers and/or (simple) mixtures of any of the abovepolymers. Certain of the above-mentioned polymers may further becrosslinked by way of standard techniques.

For the compositions of the invention in the form of gelling matrixsystems, we prefer that the principal swelling polymer that is employedis HPC, maltodextrin, scleroglucan or carboxypolymethylene, morepreferably, PEO or xanthan, and, especially, HPMC, as well as copolymersand/or (simple) mixtures of any of these polymers. Iota-carrageenan isalso preferred.

When PEO, xanthan and HPMC are employed in (that is, as at least one ofthe polymers of) the hydrophilic gelling component, preferred molecularweights (that is, weight average molecular weights, as determined bystandard techniques, such as osmometry, size-exclusion chromatographywith a refraction detector (in which molecular weight is determined byway of standard calibration curves), light scattering and/orultracentrifuge techniques), for these polymers are in the range 5,000g/mol up to 200,000,000 g/mol, such as up to 100,000,000 g/mol,preferably up to 25,000,000 g/mol and more preferably up to 20,000,000g/mol. Mixtures of PEO, xanthan and HPMC polymers with differentmolecular weights within these ranges may be employed.

Suitable HPMC polymers also include those that produce 2% w/w solutionsof polymer in water with viscosities, as measured by standardtechniques, such as those described generally in the United StatesPharmacopeia XXIV (USP XXIV/NF19) at page 2002 et seq, as well as,specifically, at pages 843 and 844 (the relevant disclosures in whichdocument are hereby incorporated by reference), of between 3 and 150,000cps (at 20° C.), such as between 10 and 120,000 cps, preferably between30 and 50,000 cps and more preferably between 50 and 15,000 cps.Mixtures of HPMC polymers with different viscosities within these rangesmay be employed, in order, for example, to produce HPMC mixtures whichproduce solutions as mentioned above with “average” viscosities (i.e. aviscosity for the mixture) within the above-mentioned preferred ranges.Similarly, mixtures of HPMC polymers (with viscosities and/or “average”viscosities within these ranges) with other above-mentioned polymers maybe employed. Suitable HPMC polymers include those fulfilling the UnitedStates Pharmacopeia standard substitution types 2208, 2906, 2910 and1828 (see USP XXIV/NF19 for further details). Suitable HPMC polymersthus include those sold under the trademark METHOCEL™ (Dow ChemicalCorporation) or the trademark METOLOSE™ (Shin-Etsu).

Suitable xanthan polymers include those that produce 1% w/w solutions ofpolymer in water with viscosities, as measured by standard techniques,such as those described generally in the United States Pharmacopeia XXIV(USP XXIV/NF19) at page 2002 et seq, as well as, specifically, at pages2537 and 2538 (the relevant disclosures in which document are herebyincorporated by reference), of between 60 and 2,000 cps (at 24° C.), forexample between 600 and 1,800 cps and preferably between 1,200 and 1,600cps. Mixtures of xanthan polymers with different viscosities withinthese ranges may be employed, in order, for example, to produce xanthanmixtures which produce solutions as mentioned above with “average”viscosities (i.e. a viscosity for the mixture) within theabove-mentioned preferred ranges. Similarly, mixtures of xanthanpolymers (with viscosities and/or “average” viscosities within theseranges) with other above-mentioned polymers may be employed. Suitablexanthan polymers include those sold under the trademarks XANTURAL™ andKELTROL™ (CPKelco), and SATIAXANE™ (Degussa, Texturant Systems).

The choice of polymer will be determined by the nature of the activeingredient/drug that is employed in the composition of the invention aswell as the desired rate of release. In particular, it will beappreciated by the skilled person, for example in the case of HPMC, thata higher molecular weight will, in general, provide a slower rate ofrelease of drug from the composition. Furthermore, in the case of HPMC,different degrees of substitution of methoxyl groups and hydroxypropoxylgroups will give rise to changes in the rate of release of drug from thecomposition. In this respect, and as stated above, it may be desirableto provide compositions of the invention in the form of gelling matrixsystems in which the polymer carrier is provided by way of a blend oftwo or more polymers of, for example, different molecular weights, forexample as described hereinafter, in order to produce a particularrequired or desired release profile.

When in the form of gelling matrix systems, we have also found that rateof release of drug from compositions of the invention may be furthercontrolled by way of controlling the drug:polymer ratio within, and thesurface area:volume ratio of, individual compositions (for exampletablets) comprising drug and polymer carrier system.

Compositions of the invention, whether in the form of a gelling matrixsystem or otherwise, may contain one or more further excipients (inaddition to the polymer carrier system) to further modify drug release,to improve the physical and/or chemical properties of the finalcomposition, and/or to facilitate the process of manufacture. Suchexcipients are conventional in the formulation of modified releasecompositions.

For example, compositions of the invention may contain one or more ofthe following diluents: calcium phosphate (monocalcium phosphate,dicalcium phosphate and tricalcium phosphate), lactose, microcrystallinecellulose, mannitol, sorbitol, titanium dioxide, aluminium silicate andthe like. Preferred diluents include microcrystalline cellulose and alsomannitol.

Compositions of the invention may contain one or more of the followinglubricants: magnesium stearate, sodium stearyl fumarate and the like.

Compositions of the invention may contain a glidant, such as a colloidalsilica.

Compositions of the invention may contain one or more of the followingbinders: polyvinylpyrrolidone, lactose, mannitol, microcrystallinecellulose, a polyethylene glycol (PEG), a HPMC of a low molecularweight, a MC of a low molecular weight, a HPC of a low molecular weightand the like. Preferred binders include microcrystalline cellulose.

Compositions of the invention may contain one or more of the followingpH controlling agents: organic acids (for example. citric acid and thelike) or alkali metal (for example sodium) salts thereof,pharmaceutically acceptable salts (for example sodium, magnesium orcalcium salts) of inorganic acids (such as carbonic acid or phosphoricacid), oxides of magnesium, as well as alkali, and alkaline earth metal(for example sodium, calcium, potassium and the like) sulphates,metabisulphates, propionates and sorbates.

Other further excipients may include colourants, flavourings,solubilising agents (such as SDS), coating agents, preservatives, etc.

Combinations of the above-stated further excipients may be employed.

It will be appreciated that some of the above mentioned furtherexcipients, which may be present in the final composition of theinvention, may have more than one of the above-stated functions.Moreover, further excipients mentioned above may also function as partof a hydrophilic gelling component in a gelling matrix system.

The total amount of further excipients (not including, in the case ofgelling matrix systems, the principal polymer carrier(s)) that may bepresent in the composition of the invention will depend upon the natureof the composition, as well as the nature, and amounts of, the otherconstituents of that composition, and may be an amount of up to 85%, forexample between 0.1 to 75%, such as 0.2 to 65%, preferably 0.3 to 55%,more preferably 0.5 to 45% and especially 1 to 40%, such as 2 to 35%w/w. In any event, the choice, and amount, of excipient(s) may bedetermined routinely (that is without recourse to inventive input) bythe skilled person.

In gelling matrix systems, the amount of polymer in the system should beenough to ensure that a sufficient dose of drug is provided over thedosing interval to produce the desired therapeutic effect. Thus, for agelling matrix system, we prefer that it takes at least 2 hours(preferably at least 4 hours, especially at least 6 hours) for 80%(especially 60%) of the initial drug content of the composition to bereleased to a patient after administration under the test conditionsdescribed hereinafter, and particularly over a period of between 8 and24 hours. Most preferably at least 80% of the initial drug content ofthe composition is released at a time somewhere between 8 and 24 hours.Suitable amounts of polymer that may be included, which will depend uponinter alia the active ingredient that is employed in the composition,any excipients that may be present and the nature of the polymer that isemployed, are in the range 5 to 99.5%, for example 10 to 95%, preferably30 to 80% w/w. In any event, the choice, and amount, of polymer may bedetermined routinely by the skilled person.

In another preferred formulation we prefer that the compounds of theinvention are formulated together in a gelling matrix compositioncomprising iota-carrageenan and one or more neutral gelling polymers.

Iota-carrageenan is preferably present in such a preferred preparationat a level of more that 15% by weight. Preferred grades ofiota-carrageenan include pharmaceutical grade iota-carrageenan (forexample, available from FMC Biopolymer), which has a viscosity of notless than 5 centipoise (cps), preferably in the range 5–10 cps (for a1.5% solution warmed to 82° C., after which the viscosity is measured at75° C. with a Brookfield LV viscometer fitted with a #1 spindle runningat a speed of 30 rpm), and technical grade iota-carrageenan (forexample, available from Fluka Biochemica), which preferably has aviscosity of not less than 14 mPa.s, for a 0.3% aqueous solution warmedto 20° C., after which the viscosity is measured using a fallingballviscometer, of type Haake, used together with a Lauda thermostat C3 andHakke Mess-System III, and using gold-coated stainless steel balls ofdensity 7.8 g/cm³.

The neutral gelling polymer may be a single, or a mixture of more thanone, neutral polymer(s) having gelling properties and havingsubstantially pH-independent solubility. The neutral gelling polymer is,preferably, present in the formulation at a level of more that 10% butpreferably more than 20% by weight.

Suitable neutral gelling polymers include polyethylene oxide (PEO),derivatives and members of the PEO family (for example, polyethyleneglycol (PEG)), preferably existing naturally in the solid state, ofsuitable molecular weight or viscosity. If used as a single neutralgelling polymer, a PEO preferably has a MW of ≧4 million (4M),corresponding to an aqueous solution viscosity range of 1650–5500 mPa.s(or 1650–5500 cps; measured for a 1% aqueous solution at 25° C., using aBrookfield RVF viscometer, with No. 2 spindle, at 2 rpm). Other examplesof suitable PEOs include a PEO of MW around 5 million (5M),corresponding to an aqueous solution viscosity range of 5500–7500 mPa.s,or a PEO MW around 8 million (8M), corresponding to an aqueous solutionviscosity range of 10000–15000 mPa.s. This range covers the value fortypical solution viscosity (in cps) measured at 25° C., quoted for thispolymer, in the USP 24/NF 19, 2000 edition, pp. 2285–2286. If PEG isused as a single neutral gelling polymer it preferably has a highmolecular weight, for example, a MW of around 20000, corresponding to aviscosity range of 2700–3500 mPa.s (or 2700–3500 cps), measured using a50% aqueous solution (w/w) at 20° C., using a capillary viscometer(Ubbelohde or equivalent). [Ref: European Pharmacopoeia 3^(rd) Ed.,2000, Supplement, pp. 908–909.]

Other suitable neutral gelling polymers include cellulose derivativessuch as hydroxypropylmethyl cellulose (HPMC) or hydroxyethylcellulose(HEC) with suitably high viscosities (for example “HPMC 50 cps”, “HPMC10000 cps”, “HPMC 15000 cps”, “HEC type HH” or “HEC type H”). When usedas a single neutral polymer, hydroxypropylmethyl cellulose polymers like“HPMC 10000 cps” and “HPMC 15000 cps” have, respectively, apparentviscosities of 7500–14000 mPa.s (or 7500–14000 cps), and 11250–21000mPa.s (or 11250–21000 cps), when measured at 20° C. with a 2% (w/w)aqueous solution, calculated with reference to the dried substance,using a capillary viscometer (Ubbelohde or equivalent). One type ofhydroxyethylcellulose polymer, for example, “Natrosol 250 Pharma, typeHH”, from Hercules Incorporated (Aqualon), shows typically a Brookfieldviscosity of about 20,000 mPa.s using a Brookfield Synchro-Lectric ModelLVF instrument, at the conditions 1% solution concentration, spindle no.4, spindle speed 30 rpm, factor 200, 25° C. (See Natrosol Physical andChemical Properties booklet, 33.007-E6 (1993), p. 21).

Particular formulations that may be mentioned include those in whichcompound of the invention is formulated together with iota-carageenanand HPMC (10,000 cps) in a 50:50 (wt %) ratio, or together withiota-carageenan and HPMC (50 cps) & HPMC (10,000 cps) in a 35:60:5 (wt%) ratio, or together with iota-carageenan and PEO 4M in a 50:50 (wt %)ratio, Preferred additional excipients in such formulations includelubricants, such as sodium stearyl fumarate.

In one aspect the invention provides a non-injectable formulation of theinvention comprising Compound A, B or C or a salt thereof; an HPMC and alubricant (such as sodium stearyl fumarate). In a further aspect theformulation may comprise a mixture of 2 or more HPMCs of differentviscosities (such as 10,000 cPs and 50 cPs). Further, the formulationmay additionally comprise a solubilising agent [such as sodium dodecylsulphate (SDS), sodium lauryl sulphate or polyoxyl 40 hydrogenatedcastor oil].

Suitable amounts of active ingredient in the compositions of theinvention, whether in the form of gelling matrix systems or otherwise,depend upon many factors, such as the nature of that ingredient (freebase/salt etc), the dose that is required, and the nature, and amounts,of other constituents of the composition. However, they may be in therange 0.5 to 80%, for example 1 to 75%, such as 3 to 70%, preferably 5to 65%, more preferably 10 to 60% and especially 15 to 55% w/w. In anyevent, the amount of active ingredient to be included may be determinedroutinely by the skilled person.

A typical daily dose of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, is in the range 0.001 to 100 mg/kg body weightof free base (that is, in the case of a salt, excluding any weightresulting from the presence of a counter ion), irrespective of thenumber of individual doses that are administered during the course ofthat day. A preferred daily dose is in the range 20–500 mg.

Compositions of the invention such as those described hereinbefore maybe made in accordance with well known techniques such as those describedin the references mentioned hereinbefore. Compositions of the inventionthat are in the form of gelling matrix systems may be prepared bystandard techniques, and using standard equipment, known to the skilledperson, including wet or dry granulation, direct compression/compaction,drying, milling, mixing, tabletting and coating, as well as combinationsof these processes, for example as described hereinafter.

Although compositions of the invention are preferably adapted to beadministered orally, their use is not limited to that mode ofadministration. Parenteral modified release compositions of theinvention, which may include systems that are well known to thoseskilled in the art, such as those based upon poloxamers, biodegradablemicrospheres, liposomes, suspensions in oils and/or emulsions, may beprepared in accordance with standard techniques, for example asdescribed by Leung et al in “Controlled Drug Delivery: Fundamentals andApplications” (Drugs and the Pharmaceutical Sciences; vol. 29), 2^(nd)edition, eds. Robinson and Lee, Dekker (1987) at Chapter 10, page 433,the disclosure in which document is hereby incorporated by reference.

The compositions of the invention may be dosed once or more times daily(preferably once, but no more than twice, daily), irrespective of thenumber of individual units (formulations/compositions) that areadministered as part of one “dose”.

The formulations of the invention are administered to mammalian patients(including humans), and, for compounds of formula (I) wherein R² is nothydrogen, are thereafter metabolised in the body to form compounds offormula (I) wherein R² is hydrogen that are pharmacologically active.

According to a further aspect of the invention there is thus provided aformulation of the invention for use as a pharmaceutical.

In particular, the compounds of formula (I) are, or are metabolisedfollowing administration to form, potent inhibitors of thrombin, forexample as may be demonstrated in the tests described in inter aliainternational patent application No. PCT/SE01/02657, as well asinternational patent applications WO 02/14270, WO 01/87879 and WO00/42059, the relevant disclosures in which documents are herebyincorporated by reference.

By “prodrug of a thrombin inhibitor”, we include compounds that aremetabolised following administration and form a thrombin inhibitor, inan experimentally-detectable amount, following administration.

By “active ingredient” and “active substance” we mean the pharmaceuticalagent (covering thrombin inhibitor and prodrugs thereof) present in theformulation.

The formulations of the invention are thus expected to be useful inthose conditions where inhibition of thrombin is required, and/orconditions where anticoagulant therapy is indicated, including thefollowing:

The treatment and/or prophylaxis of thrombosis and hypercoagulability inblood and/or tissues of animals including man. It is known thathypercoagulability may lead to thrombo-embolic diseases. Conditionsassociated with hypercoagulability and thrombo-embolic diseases whichmay be mentioned include inherited or acquired activated protein Cresistance, such as the factor V-mutation (factor V Leiden), andinherited or acquired deficiencies in antithrombin III, protein C,protein S, heparin cofactor II. Other conditions known to be associatedwith hypercoagulability and thrombo-embolic disease include circulatingantiphospholipid antibodies (Lupus anticoagulant), homocysteinemi,heparin induced thrombocytopenia and defects in fibrinolysis, as well ascoagulation syndromes (for example disseminated intravascularcoagulation (DIC)) and vascular injury in general (for example due tosurgery).

The treatment of conditions where there is an undesirable excess ofthrombin without signs of hypercoagulability, for example inneurodegenerative diseases such as Alzheimer's disease.

Particular disease states which may be mentioned include the therapeuticand/or prophylactic treatment of venous thrombosis (for example DVT) andpulmonary embolism, arterial thrombosis (e.g. in myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis), and systemic embolism usually from the atrium during atrialfibrillation (for example non-valvular atrial fibrillation) or from theleft ventricle after transmural myocardial infarction, or caused bycongestive heart failure; prophylaxis of re-occlusion (that isthrombosis) after thrombolysis, percutaneous trans-luminal angioplasty(PTA) and coronary bypass operations; the prevention of re-thrombosisafter microsurgery and vascular surgery in general.

Further indications include the therapeutic and/or prophylactictreatment of disseminated intravascular coagulation caused by bacteria,multiple trauma, intoxication or any other mechanism; anticoagulanttreatment when blood is in contact with foreign surfaces in the bodysuch as vascular grafts, vascular stents, vascular catheters, mechanicaland biological prosthetic valves or any other medical device; andanticoagulant treatment when blood is in contact with medical devicesoutside the body such as during cardiovascular surgery using aheart-lung machine or in haemodialysis; the therapeutic and/orprophylactic treatment of idiopathic and adult respiratory distresssyndrome, pulmonary fibrosis following treatment with radiation orchemotherapy, septic shock, septicemia, inflammatory responses, whichinclude, but are not limited to, edema, acute or chronic atherosclerosissuch as coronary arterial disease and the formation of atheroscleroticplaques, cerebral arterial disease, cerebral infarction, cerebralthrombosis, cerebral embolism, peripheral arterial disease, ischaemia,angina (including unstable angina), reperfusion damage, restenosis afterpercutaneous trans-luminal angioplasty (PTA) and coronary artery bypasssurgery.

The formulation of the present invention may also comprise anyantithrombotic agent(s) with a different mechanism of action to that ofthe compounds of formula (I), such as one or more of the following: theantiplatelet agents acetylsalicylic acid, ticlopidine and clopidogrel;thromboxane receptor and/or synthetase inhibitors; fibrinogen receptorantagonists; prostacyclin mimetics; phosphodiesterase inhibitors;ADP-receptor (P₂T) antagonists; and inhibitors of carboxypeptidase U(CPU).

Compounds of formula (I) that inhibit trypsin and/or thrombin may alsobe useful in the treatment of pancreatitis.

The formulations of the invention are thus indicated both in thetherapeutic and/or prophylactic treatment of these conditions.

The formulations of the invention are useful in the delivery of acompound of formula (I) or a salt thereof to a patient. As the compoundsof formula (I), and salts thereof, are useful in both the prophylaxisand the treatment of thrombisis, the formulations of the invention arealso useful in the treatment of such a disorder.

According to a further aspect of the invention, there is provided amethod of treatment of thrombosis which method comprises administrationof a formulation of the invention to a person suffering from, orsusceptible to, such a condition.

In a still further aspect the present invention provides a formulationof the invention in the manufacture of a medicament for use in thetreatment of thrombosis.

For the avoidance of doubt, by “treatment” we include the therapeutictreatment, as well as the prophylaxis, of a condition.

The compositions of the invention have the advantage that they mayprovide a modified release of the compounds of formula (I) or apharmaceutically acceptable salt of any of these compounds, in order toobtain a more even and/or prolonged effect against thrombosis and maythus provide efficient dosing of active ingredient preferably no morethan once or twice daily.

Compositions of the invention may also have the advantage that they maybe prepared using established pharmaceutical processing methods andemploy materials that are approved for use in foods or pharmaceuticalsor of like regulatory status.

Compounds of formula (I) can be prepared using the following procedures.

General Procedures

TLC was performed on silica gel. Chiral HPLC analysis was performedusing a 46 mm×250 mm Chiralcel OD column with a 5 cm guard column. Thecolumn temperature was maintained at 35° C. A flow rate of 1.0 mL/minwas used. A Gilson 115 UV detector at 228 nm was used. The mobile phaseconsisted of hexanes, ethanol and trifluroacetic acid and theappropriate ratios are listed for each compound. Typically, the productwas dissolved in a minimal amount of ethanol and this was diluted withthe mobile phase.

In Preparations A to I below, LC-MS/MS was performed using a HP-1100instrument equipped with a CTC-PAL injector and a 5 Tm, 4×100 mmThermoQuest, Hypersil BDS-C18 column. An API-3000 (Sciex) MS detectorwas used. The flow rate was 1.2 mL/min and the mobile phase (gradient)consisted of 10–90% acetonitrile with 90–10% of 4 mM aq. ammoniumacetate, both containing 0.2% formic acid. Otherwise, low resolutionmass spectra (LRMS) were recorded using a Micromass ZQ spectrometer inESI posneg switching ion mode (mass range m/z 100–800); and highresolution mass spectra (HRMS) were recorded using a Micromass LCTspectrometer in ES negative ionization mode (mass range m/z 100–1000)with Leucine Enkephalin (C₂₈H₃₇N₅O₇) as internal mass standard.

¹H NMR spectra were recorded using tetramethylsilane as the internalstandard.

Processes for the synthesis of compounds of formula (I) are contained inInternational Patent Application No. PCT/SE01/02657 (WO 02/44145,earliest priority date 1 Dec. 2000, filed 30 Nov. 2001, published 6 Jun.2002)), relevant information from which is incorporated herein.

PREPARATION A Preparation of Compound A

(i) 3-Chloro-5-methoxybenzaldehyde

3,5-Dichloroanisole (74.0 g, 419 mmol) in THF (200 mL) was addeddropwise to magnesium metal (14.2 g, 585 mmol, pre-washed with 0.5 NHCl) in THF (100 mL) at 25° C. After the addition, 1,2-dibromoethane(3.9 g, 20.8 mmol) was added dropwise. The resultant dark brown mixturewas heated at reflux for 3 h. The mixture was cooled to 0° C., andN,N-dimethylformamide (60 mL) was added in one portion. The mixture waspartitioned with diethyl ether (3×400 mL) and 6N HCl (500 mL). Thecombined organic extracts were washed with brine (300 mL), dried(Na₂SO₄), filtered and concentrated in vacuo to give an oil. Flashchromatography (2×) on silica gel eluting with Hex:EtOAc (4:1) affordedthe sub-title compound (38.9 g, 54%) as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 9.90 (s, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 7.15(s, 1H), 3.87 (s, 3H).

(ii) 3-Chloro-5-hydroxybenzaldehyde

A solution of 3-chloro-5-methoxybenzaldehyde (22.8 g, 134 mmol; see step(i) above) in CH₂Cl₂ (250 mL) was cooled to 0° C. Boron tribromide (15.8mL, 167 mmol) was added dropwise over 15 min. After stirring, thereaction mixture for 2 h, H₂O (50 mL) was added slowly. The solution wasthen extracted with Et₂O (2×100 mL). The organic layers were combined,dried (Na₂SO₄), filtered and concentrated in vacuo. Flash chromatographyon silica gel eluting with Hex:EtOAc (4:1) afforded the sub-titlecompound (5.2 g, 25%).

¹H NMR (300 MHz, CDCl₃) δ 9.85 (s, 1H), 7.35 (s, 1H), 7.20 (s, 1H), 7.10(s, 1H), 3.68 (s, 1H)

(iii) 3-Chloro-5-difluoromethoxybenzaldehyde

A solution of 3-chloro-5-hydroxybenzaldehyde (7.5 g, 48 mmol; see step(ii) above) in 2-propanol (250 mL) and 30% KOH (100 mL) was heated toreflux. While stirring, CHClF₂ was bubbled into the reaction mixture for2 h. The reaction mixture was cooled, acidified with 1N HCl andextracted with EtOAc (2×100 mL). The organics were washed with brine(100 mL), dried (Na₂SO₄), filtered and concentrated in vacuo. Flashchromatography on silica gel eluting with Hex:EtOAc (4:1) afforded thesub-title compound (4.6 g, 46%).

¹H NMR (300 MHz, CDCl₃) γ 9.95 (s, 1H), 7.72 (s, 1H), 7.52 (s, 1H), 7.40(s, 1H), 6.60 (t, J_(H-F)=71.1 Hz, 1H)

(iv) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OTMS)CN

A solution of 3-chloro-5-difluoromethoxybenzaldehyde (4.6 g, 22.3 mmol;see step (iii) above) in CH₂Cl₂ (200 mL) was cooled to 0° C. ZnI₂ (1.8g, 5.6 mmol) and trimethylsilyl cyanide (2.8 g, 27.9 mmol) were addedand the reaction mixture was allowed to warm to room temperature andstirred for 15 h. The mixture was partially concentrated in vacuoyielding the sub-title compound as a liquid, which was used directly instep (v) below without further purification or characterization.

(v) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(NH)OEt

Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OTMS)CN (6.82 g, assume 22.3 mmol; see step(iv) above) was added dropwise to HCl/EtOH (500 mL). The reactionmixture was stirred 15 h, then partially concentrated in vacuo yieldingthe sub-title compound as a liquid, which was used in step (vi) withoutfurther purification or characterization.

(vi) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OEt

Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(NH)OEt (6.24 g, assume 22.3 mmol; seestep (v) above) was dissolved in THF (250 mL), 0.5M H₂SO₄ (400 mL) wasadded and the reaction was stirred at 40° C. for 65 h, cooled and thenpartially concentrated in vacuo to remove most of the THF. The reactionmixture was then extracted with Et₂O (3×100 mL), dried (Na₂SO₄),filtered and concentrated in vacuo to afford the sub-title compound as asolid, which was used in step (vii) without further purification orcharacterization.

(vii) Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OH

A solution of Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OEt (6.25 g, assume 22.3mmol; see step (vi) above) in 2-propanol (175 mL) and 20% KOH (350 mL)was stirred at room temperature 15 h. The reaction was then partiallyconcentrated in vacuo to remove most of the 2-propanol. The remainingmixture was acidified with 1M H₂SO₄, extracted with Et₂O (3×100 mL),dried (Na₂SO₄) and concentrated in vacuo to give a solid. Flashchromatography on silica gel eluting with CHCl₃:MeOH:concentrated NH₄OH(6:3:1) afforded the ammonium salt of the sub-title compound. Theammonium salt was then dissolved in a mixture of EtOAc (75 mL) and H₂O(75 mL) and acidified with 2N HCl. The organic layer was separated andwashed with brine (50 mL), dried (Na₂SO₄) and concentrated in vacuo toafford the sub-title compound (3.2 g, 57% from steps (iv) to (vii)).

¹H NMR (300 MHz, CD₃OD) δ 7.38 (s, 1H), 7.22 (s, 1H), 7.15 (s, 1H), 6.89(t, J_(H-F)=71.1 Hz, 1H), 5.16 (s, 1H)

(viii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (a) andPh(3-Cl)(5-OCHF₂)—(S)CH(OAc)C(O)OH (b)

A mixture of Ph(3-Cl)(5-OCHF₂)—(R,S)CH(OH)C(O)OH (3.2 g, 12.7 mmol; seestep (vii) above) and Lipase PS “Amano” (˜2.0 g) in vinyl acetate (125mL) and MTBE (125 mL) was heated at reflux for 48 h. The reactionmixture was cooled, filtered through Celite® and the filter cake washedwith EtOAc. The filtrate was concentrated in vacuo and subjected toflash chromatography on silica gel eluting with CHCl₃:MeOH:concentratedNH₄OH (6:3:1) yielding the ammonium salts of the sub-title compounds (a)and (b). Compound (a) as a salt was dissolved in H₂O, acidified with 2NHCl and extracted with EtOAc. The organic layer was washed with brine,dried (Na₂SO₄), filtered and concentrated in vacuo to afford thesub-title compound (a) (1.2 g, 37%).

For sub-title compound (a)

¹H NMR (300 MHz, CD₃OD) δ 7.38 (s, 1H), 7.22 (s, 1H), 7.15 (s, 1H), 6.89(t, J_(H-F)=71.1 Hz, 1H), 5.17 (s, 1H)

(ix) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc)

To a solution of Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (1.1 g, 4.4 mmol; seestep (viii) above) and H-Aze-Pab(Teoc) (see international patentapplication WO 00/42059, 2.6 g, 5.7 mmol) in DMF (50 mL) at 0° C. wasadded PyBOP (2.8 g, 5.3 mmol) and collidine (1.3 g, 10.6 mmol). Thereaction was stirred at 0° C. for 2 h and then at room temperature foran additional 15 h. The reaction mixture was concentrated in vacuo andflash chromatographed on silica gel (3×), eluting first with CHCl₃:EtOH(9:1), then with EtOAc:EtOH (20:1) and finally eluting with CH₂Cl₂:CH₃OH(95:5) to afford the sub-title compound (1.0 g, 37%) as a white solid.

¹H NMR (300 MHz, CD₃OD, mixture of rotamers) δ 7.79–7.85 (d, J=8.7 Hz,2H), 7.15–7.48 (m, 5H), 6.89 and 6.91 (t, J_(H-F)=71.1 Hz, 1H), 5.12 and5.20 (s, 1H), 4.75–4.85 (m, 1H), 3.97–4.55 (m, 6H), 2.10–2.75 (m, 2H),1.05–1.15 (m, 2H), 0.09 (s, 9H)

MS (m/z) 611 (M+1)⁺

(x) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OMe, Teoc)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.40 g, 0.65 mmol; seestep (ix) above), was dissolved in 20 mL of acetonitrile and 0.50 g (6.0mmol) of O-methyl hydroxylamine hydrochloride was added. The mixture washeated at 70° C. for 2 h. The solvent was evaporated and the residue waspartitioned between water and ethyl acetate. The aqueous phase wasextracted twice more with ethyl acetate and the combined organic phasewas washed with water, brine, dried (Na₂SO₄), filtered and evaporated.Yield: 0.41 g (91%).

¹H-NMR (400 MHz; CDCl₃): δ 7.83 (bt, 1H), 7.57 (bs, 1H), 7.47 (d, 2H),7.30 (d, 2H), 7.20 (m, 1H), 7.14 (m, 1H), 7.01 (m, 1H), 6.53 (t, 1H),4.89 (s, 1H), 4.87 (m, 1H), 4.47 (m, 2H), 4.4–4.2 (b, 1H), 4.17–4.1 (m,3H), 3.95 (s, 3H), 3.67 (m, 1H), 2.68 (m, 1H), 2.42 (m, 1H) 0.97 (m,2H), 0.01 (s, 9H).

(xi) Compound A

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OMe, Teoc) (0.40 g, 0.62 mmol;see step (x) above), was dissolved in 5 mL of TFA and allowed to reactfor 30 min. TFA was evaporated and the residue was partitioned betweenethyl acetate and NaHCO₃ (aq.). The aqueous phase was extracted twicemore with ethyl acetate and the combined organic phase was washed withwater, brine, dried (Na₂SO₄), filtered and evaporated. The product wasfreeze dried from water/acetonitrile. No purification wasnecessary.Yield: 0.28 g (85%).

¹H-NMR (600 MHz; CDCl₃): δ 7.89 (bt, 1H), 7.57 (d, 2H), 7.28 (d, 2H),7.18 (m, 1H), 7.13 (m, 1H), 6.99 (m, 1H), 6.51 (t, 1H), 4.88 (s, 1H),4.87 (m, 1H), 4.80 (bs, 2H), 4.48 (dd, 1H), 4.43 (dd, 1H), 4.10 (m, 1H),3.89 (s, 3H), 3.68 (m, 1H), 2.68 (m, 1H), 2.40 (m, 1H).

¹³C-NMR (125 MHz; CDCl₃): (carbonyl and/or amidine carbons, rotamers) δ172.9, 170.8, 152.7, 152.6

HRMS calculated for C₂₂H₂₃ClF₂N₄O₅ (M−H)⁻ 495.1242, found 495.1247

PREPARATION B Preparation of Compound B

(i) 2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile

(Methylsulfinyl)(methylthio)methane (7.26 g, 0.0584 mol) was dissolvedin 100 mL of dry THF under argon and was cooled to −78° C. Butyllithiumin hexane (16 mL 1.6M, 0.0256 mol) was added dropwise with stirring. Themixture was stirred for 15 min. Meanwhile, a solution of3,4,5-trifluorobenzonitrile (4.0 g, 0.025 mmol) in 100 mL of dry THF wascooled to −78° C. under argon and the former solution was added througha cannula to the latter solution over a period of 35 min. After 30 min,the cooling bath was removed and when the reaction had reached roomtemperature it was poured into 400 mL of water. The THF was evaporatedand the remaining aqueous layer was extracted three times with diethylether. The combined ether phase was washed with water, dried (Na₂SO₄)and evaporated. Yield: 2.0 g (30%).

¹H NMR (500 MHz, CDCl₃) δ 7.4–7.25 (m, 2H), 5.01 (s, 1H, diasteromer),4.91 (s, 1H, diasteromer), 2.88 (s, 3H, diasteromer), 2.52 (s, 3H,diasteromer), 2.49 (s, 3H, diasteromer), 2.34 (s, 3H, diasteromer), 1.72(broad, 1H)

(ii) 2,6-Difluoro-4-formylbenzonitrile

2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile (2.17 g,8.32 mmol; see step (i) above) was dissolved in 90 mL of THF and 3.5 mLof concentrated sulfuric acid was added. The mixture was left at roomtemperature for 3 days and subsequently poured into 450 mL of water.Extraction three times with EtOAc followed and the combined etherealphase was washed twice with aqueous sodium bicarbonate and with brine,dried (Na₂SO₄) and evaporated. Yield: 1.36 g (98%). The position of theformyl group was established by ¹³C NMR. The signal from the fluorinatedcarbons at 162.7 ppm exhibited the expected coupling pattern with twocoupling constants in the order of 260 Hz and 6.3 Hz respectivelycorresponding to an ipso and a meta coupling from the fluorine atoms.

¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 7.33 (m, 2H)

(iii) 2.6-Difluoro-4-hydroxymethylbenzonitrile

2,6-Difluoro-4-formylbenzonitrile (1.36 g, 8.13 mmol; see step (ii)above) was dissolved in 25 mL of methanol and cooled on an ice bath.Sodium borohydride (0.307 g, 8.12 mmol) was added in portions withstirring and the reaction was left for 65 min. The solvent wasevaporated and the residue was partitioned between diethyl ether andaqueous sodium bicarbonate. The ethereal layer was washed with moreaqueous sodium bicarbonate and brine, dried (Na₂SO₄) and evaporated. Thecrude product crystallised soon and could be used without furtherpurification. Yield: 1.24 g (90%).

¹H NMR (400 MHz, CDCl₃) δ 7.24 (m, 2H), 4.81 (s, 2H), 2.10 (broad, 1H)

(iv) 4-Cyano-2,6-difluorobenzyl methanesulfonate

To an ice cooled solution of 2,6-difluoro-4-hydroxymethylbenzonitrile(1.24 g, 7.32 mmol; see step (iii) above) and methanesulfonyl chloride(0.93 g, 8.1 mmol) in 60 mL of methylene chloride was addedtriethylamine (0.81 g, 8.1 mmol) with stirring. After 3 h at 0° C., themixture was washed twice with 1M HCl and once with water, dried (Na₂SO₄)and evaporated. The product could be used without further purification.Yield: 1.61 g (89%).

¹H NMR (300 MHz, CDCl₃) δ 7.29 (m, 2H), 5.33 (s, 2H), 3.07 (s, 3H)

(v) 4-Azidomethyl-2,6-difluorobenzonitrile

A mixture of 4-cyano-2,6-difluorobenzyl methanesulfonate (1.61 g, 6.51mmol; see step (iv) above) and sodium azide (0.72 g, 0.0111 mol) in 10mL of water and 20 mL of DMF was stirred at room temperature overnight.The resultant was subsequently poured into 200 mL of water and extractedthree times with diethyl ether. The combined ethereal phase was washedfive times with water, dried (Na₂SO₄) and evaporated. A small sample wasevaporated for NMR purposes and the product crystallised. The rest wasevaporated cautiously but not until complete dryness. Yield(theoretically 1.26 g) was assumed to be almost quantitative based onNMR and analytical HPLC.

¹H NMR (400 MHz, CDCl₃) δ 7.29 (m, 2H), 4.46 (s, 2H)

(vi) 4-Aminomethyl-2,6-difluorobenzonitrile

This reaction was carried out according to the procedure described in J.Chem. Res. (M) (1992) 3128. To a suspension of 520 mg of 10% Pd/C (50%moisture) in 20 mL of water was added a solution of sodium borohydride(0.834 g, 0.0221 mol) in 20 mL of water. Some gas evolution resulted.4-Azidomethyl-2,6-difluorobenzonitrile (1.26 g, 6.49 mmol; see step (v)above) was dissolved in 50 mL of THF and added to the aqueous mixture onan ice bath over 15 min. The mixture was stirred for 4 h, whereafter 20mL of 2M HCl was added and the mixture was filtered through Celite. TheCelite was rinsed with more water and the combined aqueous phase waswashed with EtOAc and subsequently made alkaline with 2M NaOH.Extraction three times with methylene chloride followed and the combinedorganic phase was washed with water, dried (Na₂SO₄) and evaporated.Yield: 0.87 g (80%).

¹H NMR (400 MHz, CDCl₃) δ 7.20 (m, 2H), 3.96 (s, 2H), 1.51 (broad, 2H)

(vii) 2,6-Difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile

A solution of 4-aminomethyl-2,6-difluorobenzonitrile (0.876 g, 5.21mmol; see step (vi) above) was dissolved in 50 mL of THF anddi-tert-butyl dicarbonate (1.14 g, 5.22 mmol) in 10 mL of THF was added.The mixture was stirred for 3.5 h. The THF was evaporated and theresidue was partitioned between water and EtOAc. The organic layer waswashed three times with 0.5 M HCl and water, dried (Na₂SO₄) andevaporated. The product could be used without further purification.Yield: 1.38 g (99%).

¹H NMR (300 MHz, CDCl₃) δ 7.21 (m, 2H), 4.95 (broad, 1H), 4.43 (broad,2H), 1.52 (s, 9H)

(viii) Boc-Pab(2,6-diF)(OH)

A mixture of 2,6-difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile(1.38 g, 5.16 mmol; see step (vii) above), hydroxylamine hydrochloride(1.08 g, 0.0155 mol) and triethylamine (1.57 g, 0.0155 mol) in 20 mL ofethanol was stirred at room temperature for 36 h. The solvent wasevaporated and the residue was partitioned between water and methylenechloride. The organic layer was washed with water, dried (Na₂SO₄) andevaporated. The product could be used without further purification.Yield: 1.43 g (92%).

¹H NMR (500 MHz, CD₃OD) δ 7.14 (m, 2H), 4.97 (broad, 1H), 4.84 (broad,2H), 4.40 (broad, 2H), 1.43 (s, 9H)

(ix) Boc-Pab(2,6-diF)×HOAc

This reaction was carried out according to the procedure described byJudkins et al, Synth. Comm. (1998) 4351. Boc-Pab(2,6-diF)(OH) (1.32 g,4.37 mmol; see step (viii) above), acetic anhydride (0.477 g, 4.68 mmol)and 442 mg of 10% Pd/C (50% moisture) in 100 mL of acetic acid washydrogenated at 5 atm pressure for 3.5 h. The mixture was filteredthrough Celite, rinsed with ethanol and evaporated. The residue wasfreeze-dried from acetonitrile and water and a few drops of ethanol. Thesub-title product could be used without further purification. Yield:1.49 g (99%).

¹H NMR (400 MHz, CD₃OD) δ 7.45 (m, 2H), 4.34 (s, 2H), 1.90 (s, 3H), 1.40(s, 9H)

(x) Boc-Pab(2,6-diF)(Teoc)

To a solution of Boc-Pab(2,6-diF)×HOAc (1.56 g, 5.49 mmol; see step (ix)above) in 100 mL of THF and 1 mL of water was added2-(trimethylsilyl)ethyl p-nitrophenyl carbonate (1.67 g, 5.89 mmol). Asolution of potassium carbonate (1.57 g, 0.0114 mol) in 20 mL of waterwas added dropwise over 5 min. The mixture was stirred overnight. TheTHF was evaporated and the residue was partitioned between water andmethylene chloride. The aqueous layer was extracted with methylenechloride and the combined organic phase was washed twice with aqueoussodium bicarbonate, dried (Na₂SO₄) and evaporated. Flash chromatographyon silica gel with heptane/EtOAc=2/1 gave 1.71 g (73%) of pure compound.

¹H NMR (400 MHz, CDCl₃) δ 7.43 (m, 2H), 4.97 (broad, 1H), 4.41 (broad,2H), 4.24 (m, 2H), 1.41 (s, 9H), 1.11 (m, 2H), 0.06 (s, 9H)

(xi) Boc-Aze-Pab(2,6-diF)(Teoc)

Boc-Pab(2,6-diF)(Teoc) (1.009 g, 2.35 mmol; see step (x) above) wasdissolved in 50 mL of EtOAc saturated with HCl(g). The mixture was leftfor 10 min., evaporated and dissolved in 18 mL of DMF, and then cooledon an ice bath. Boc-Aze-OH (0.450 g, 2.24 mmol), PyBOP (1.24 g, 2.35mmol) and lastly diisopropylethyl amine (1.158 g, 8.96 mmol) were added.The reaction mixture was stirred for 2 h and then poured into 350 mL ofwater and extracted three times with EtOAc. The combined organic phasewas washed with brine, dried (Na₂SO₄) and evaporated. Flashchromatography on silica gel with heptane:EtOAc (1:3) gave 1.097 g (96%)of the desired compound.

¹H NMR (500 MHz, CDCl₃) δ 7.46 (m, 2H), 4.65–4.5 (m, 3H), 4.23 (m, 2H),3.87 (m, 1H), 3.74 (m, 1H), 2.45–2.3 (m, 2H), 1.40 (s, 9H), 1.10 (m,2H), 0.05 (s, 9H)

(xii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc)

Boc-Aze-Pab(2,6-diF)(Teoc) (0.256 g, 0.500 mmol; see step (xi) above)was dissolved in 20 mL of EtOAc saturated with HCl(g). The mixture wasleft for 10 min. and evaporated and dissolved in 5 mL of DMF.Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (0.120 g, 0.475 mmol; see PreparationA(viii) above), PyBOP (0.263 g, 0.498 mmol) and lastly diisopropylethylamine (0.245 g, 1.89 mmol) were added. The reaction mixture was stirredfor 2 h and then poured into 350 mL of water and extracted three timeswith EtOAc. The combined organic phase was washed with brine, dried(Na₂SO₄) and evaporated. Flash chromatography on silica gel with EtOAcgave 0.184 g (60%) of the desired sub-title compound.

¹H NMR (400 MHz, CD₃OD, mixture of rotamers) δ 7.55–7.45 (m, 2H), 7.32(m, 1H, major rotamer), 7.27 (m, 1H, minor rotamer), 7.2–7.1 (m, 2H),6.90 (t, 1H, major rotamer), 6.86 (t, 1H, minor rotamer), 5.15 (s, 1H,major rotamer), 5.12 (m, 1H, minor rotamer), 5.06 (s, 1H, minorrotamer), 4.72 (m, 1H, major rotamer), 4.6–4.45 (m, 2H), 4.30 (m, 1H,major rotamer), 4.24 (m, 2H), 4.13 (m, 1H, major rotamer), 4.04 (m, 1H,minor rotamer), 3.95 (m, 1H, minor rotamer), 2.62 (m, 1H, minorrotamer), 2.48 (m, 1H, major rotamer), 2.22 (m, 1H, major rotamer), 2.10(m, 1H, minor rotamer), 1.07 (m, 2H), 0.07 (m, 9H)

(xiii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(OMe,Teoc)

A mixture of Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc) (64mg, 0.099 mmol; see step (xii) above) and O-methyl hydroxylaminehydrochloride (50 mg, 0.60 mmol) in 4 mL of acetonitrile was heated at70° C. for 3 h. The solvent was evaporated and the residue waspartitioned between water and EtOAc. The aqueous layer was extractedtwice with EtOAc and the combined organic phase was washed with water,dried (Na₂SO₄) and evaporated. The product could be used without furtherpurification. Yield: 58 mg (87%).

¹H NMR (400 MHz, CDCl₃) δ 7.90 (bt, 1H), 7.46 (m, 1H), 7.25–6.95 (m,5H), 6.51 (t, 1H), 4.88 (s, 1H), 4.83 (m, 1H), 4.6–4.5 (m, 2H), 4.4–3.9(m, 4H), 3.95 (s, 3H), 3.63 (m, 1H), 2.67 (m, 1H), 2.38 (m, 1H), 1.87(broad, 1H), 0.98 (m, 2H), 0.01 (s, 9H)

(xiv) Compound B

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(OMe,Teoc) (58 mg, 0.086mmol; see step (xiii) above) was dissolved in 3 mL of TFA, cooled on anice bath and allowed to react for 2 h. The TFA was evaporated and theresidue dissolved in EtOAc. The organic layer was washed twice withaqueous sodium carbonate and water, dried (Na₂SO₄) and evaporated. Theresidue was freeze-dried from water and acetonitrile to give 42 mg (92%)of the title compound.

¹H NMR (300 MHz, CDCl₃) δ 7.95 (bt, 1H), 7.2–7.1 (m, 4H), 6.99 (m, 1H),6.52 (t, 1H), 4.88 (s, 1H), 4.85–4.75 (m, 3H), 4.6–4.45 (m, 2H), 4.29(broad, 1H), 4.09 (m, 1H), 3.89 (s, 3H), 3.69 (m, 1H), 2.64 (m, 1H),2.38 (m, 1H), 1.85 (broad, 1H)

¹³C-NMR (100 MHz; CDCl₃): (carbonyl and/or amidine carbons) δ 172.1,169.8, 151.9

APCI-MS: (M+1)=533/535 m/z

PREPARATION C Preparation of Compound C

(i) (2-Monofluoroethyl) methanesulfonate

To a magnetically stirred solution of 2-fluoroethanol (5.0 g, 78.0 mmol)in CH₂Cl₂ (90 mL) under nitrogen at 0° C. was added triethylamine (23.7g, 234 mmol) and methanesulfonyl chloride (10.7 g, 93.7 mmol). Themixture was stirred at 0° C. for 1.5 h, diluted with CH₂Cl₂ (100 mL) andwashed with 2N HCl (100 mL). The aqueous layer was extracted with CH₂Cl₂(50 mL) and the combined organic extracts washed with brine (75 mL),dried (Na₂SO₄), filtered and concentrated in vacuo to afford thesub-title compound (9.7 g, 88%) as a yellow oil which was used withoutfurther purification.

¹H NMR (300 MHz, CDCl₃) δ 4.76 (t, J=4 Hz, 1H), 4.64 (t, J=4 Hz, 1H),4.52 (t, J=4 Hz, 1H), 4.43 (t, J=4 Hz, 1H), 3.09 (s, 3H).

(ii) 3-Chloro-5-monofluoroethoxybenzaldehyde

To a solution of 3-chloro-5-hydroxybenzaldehyde (8.2 g, 52.5 mmol; seePreparation A(ii) above) and potassium carbonate (9.4 g, 68.2 mmol) inDMF (10 mL) under nitrogen was added a solution of (2-monofluoroethyl)methanesulfonate (9.7 g, 68.2 mmol; see step (i) above) in DMF (120 mL)dropwise at room temperature. The mixture was heated to 100° C. for 5 hand then stirred overnight at room temperature. The reaction was cooledto 0° C., poured into ice-cold 2N HCl and extracted with EtOAc. Thecombined organic extracts were washed with brine, dried (Na₂SO₄),filtered and concentrated in vacuo. The brown oil was chromatographed onsilica gel eluting with Hex:EtOAc (4:1) to afford the sub-title compound(7.6 g, 71%) as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 9.92 (s, 1H), 7.48 (s, 1H), 7.32 (s, 1H), 7.21(s, 1H), 4.87 (t, J=4 Hz, 1H), 4.71 (t, J=3 Hz, 1H), 4.33 (t, J=3 Hz,1H), 4.24 (t, J=3 Hz, 1H).

(iii) Ph(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OTMS)CN

To a solution of 3-chloro-5-monofluoroethoxybenzaldehyde (7.6 g, 37.5mmol; see step (ii) above) and zinc iodide (3.0 g, 9.38 mmol) in CH₂Cl₂(310 mL) was added trimethylsilyl cyanide (7.4 g, 75.0 mmol) dropwise at0° C. under nitrogen. The mixture was stirred at 0° C. for 3 h and atroom temperature overnight. The reaction was diluted with H₂O (300 mL),the organic layer was separated, dried (Na₂SO₄), filtered andconcentrated in vacuo to afford the sub-title compound (10.6 g, 94%) asa brown oil that was used without further purification orcharacterisation.

(iv) Ph(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OH)C(O)OH

Concentrated hydrochloric acid (100 mL) was added toPh(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OTMS)CN (10.6 g, 5.8 mmol; see step (iii)above) and the solution stirred at 100° C. for 3 h. After cooling toroom temperature, the reaction was further cooled to 0° C., basifiedslowly with 3N NaOH (˜300 mL) and washed with Et₂O (3×200 mL). Theaqueous layer was acidified with 2N HCl (80 mL) and extracted with EtOAc(3×300 mL). The combined EtOAc extracts were dried (Na₂SO₄), filteredand concentrated in vacuo to afford the sub-title compound (8.6 g, 98%)as a pale yellow solid that was used without further purification.

R_(f)=0.28 (90:8:2 CHCl₃:MeOH:concentrated NH₄OH)

¹H NMR (300 MHz, CD₃OD) δ 7.09 (s, 1H), 7.02 (s, 1H), 6.93 (s, 1H), 5.11(s, 1H), 4.77–4.81 (m, 1H), 4.62–4.65 (m, 1H), 4.25–4.28 (m, 1H),4.15–4.18 (m, 1H).

(v) Ph(3-Cl)(5-OCH₂CH₂F)—(S)CH(OAc)C(O)OH (a) andPh(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)OH (b)

A solution of Ph(3-Cl)(5-OCH₂CH₂F)—(R,S)CH(OH)C(O)OH (8.6 g, 34.5 mmol;see step (iv) above) and Lipase PS “Amano” (4.0 g) in vinyl acetate (250mL) and MTBE (250 mL) was heated at 70° C. under nitrogen for 3 d. Thereaction was cooled to room temperature and the enzyme removed byfiltration through Celite®. The filter cake was washed with EtOAc andthe filtrate concentrated in vacuo. Chromatography on silica gel elutingwith CHCl₃:MeOH:Et₃N (90:8:2) afforded the triethylamine salt ofsub-title compound (a) as a yellow oil. In addition, the triethylaminesalt of sub-title compound (b) (4.0 g) was obtained. The salt ofsub-title compound (b) was dissolved in H₂O (250 mL), acidified with 2NHCl and extracted with EtOAc (3×200 mL). The combined organic extractswere dried (Na₂SO₄), filtered and concentrated in vacuo to yield thesub-title compound (b) (2.8 g, 32%) as a yellow oil.

Data for Sub-Title Compound (b):

R_(f)=0.28 (90:8:2 CHCl₃:MeOH:concentrated NH₄0H)

¹H NMR (300 MHz, CD₃OD) δ 7.09 (s, 1H), 7.02 (s, 1H), 6.93 (s, 1H), 5.11(s, 1H), 4.77–4.81 (m, 1H), 4.62–4.65 (m, 1H), 4.25–4.28 (m, 1H),4.15–4.18 (m, 1H).

(vi) Compound C

To a solution of Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)OH (818 mg, 3.29mmol; see step (v) above) in DMF (30 mL) under nitrogen at 0° C. wasadded HAze-Pab(OMe).2HCl (1.43 g, 4.27 mmol, see international patentapplication WO 00/42059), PyBOP (1.89 g, 3.68 mmol), and DIPEA (1.06 g,8.23 mmol). The reaction was stirred at 0° C. for 2 h and then at roomtemperature overnight. The mixture was concentrated in vacuo and theresidue chromatographed two times on silica gel, eluting first withCHCl₃:EtOH (15:1) and second with EtOAc:EtOH (20:1) to afford the titlecompound (880 mg, 54%).

R_(f)=0.60 (10:1 CHCl₃:EtOH)

¹H NMR (300 MHz, CD₃OD, complex mixture of rotamers) δ 7.58–7.60 (d, J=8Hz, 2H), 7.34 (d, J=7 Hz, 2H), 7.05–7.08 (m, 2H), 6.95–6.99 (m, 1H),5.08–5.13 (m, 1H), 4.77–4.82 (m, 1H), 4.60–4.68 (m, 1H), 3.99–4.51 (m,7H), 3.82 (s, 3H), 2.10–2.75 (m, 2H).

¹³C-NMR (150 MHz; CD₃OD): (carbonyl and/or amidine carbons) δ 173.3,170.8, 152.5.

APCI-MS: (M+1)=493 m/z.

Preparation of Compound D (Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab)

Compound D

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.045 g, 0.074 mmol; seePreparation A (ix) above), was dissolved in 3 mL of TFA and allowed toreact for 1 h. TFA was evaporated and the residue was freeze dried fromwater/acetonitrile to yield 0.043 g (100%) of the sub-title compound asits TFA salt.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.8–7.75 (m, 2H), 7.55–7.5 (m, 2H),7.35 (m, 1H, major rotamer), 7.31 (m, 1H, minor rotamer), 7.19 (m, 1H,major rotamer), 7.15 (m, 1H), 7.12 (m, 1H, minor rotamer), 6.89 (t, 1H,major rotamer), 6.87 (t, 1H, minor rotamer), 5.22 (m, 1H, minorrotamer), 5.20 (s, 1H, major rotamer), 5.13 (s, 1H, minor rotamer), 4.80(m, 1H, major rotamer), 4.6–4.4 (m, 2H), 4.37 (m, 1H, major rotamer),4.19 (m, 1H, major rotamer), 4.07 (m, 1H, minor rotamer), 3.98 (m, 1H,minor rotamer), 2.70 (m, 1H, minor rotamer), 2.55 (m, 1H, majorrotamer), 2.29 (m, 1H, major rotamer), 2.15 (m, 1H, minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidine carbons, rotamers) δ172.6, 172.5, 172.0, 171.7, 167.0

MS (m/z) 465 (M−1)⁻, 467 (M+1)⁺

Preparation of Compound E(Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF))

Compound E

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc) (81 mg, 0.127mmol; see Preparation B (xii) above) was dissolved in 0.5 mL ofmethylene chloride and cooled on an ice bath. TFA (3 mL) was added andthe reaction was left for 75 min. The TFA was evaporated and the residuewas freeze dried from water and acetonitrile. The crude product waspurified by preparative RPLC with CH₃CN:0.1M NH₄OAc (35:65) to produce39 mg (55%) of the title compound as its HOAc salt, purity: 99%.

¹H NMR (400 MHz, CD₃OD mixture of rotamers) δ 7.5–7.4 (m, 2H), 7.32 (m,1H, major rotamer), 7.28 (m, 1H, minor rotamer), 7.2–7.1 (m, 3H) 6.90(t, 1H, major rotamer), 6.86 (t, minor rotamer), 5.15 (s, 1H, majorrotamer), 5.14 (m, 1H, minor rotamer), 5.07 (s, 1H, minor rotamer), 4.72(m, 1H, major rotamer), 4.65–4.45 (m, 2H), 4.30 (m, 1H, major rotamer),4.16 (m, 1H, major rotamer), 4.03 (m, 1H, minor rotamer), 3.95 (m, 1H,minor rotamer), 2.63 (m, 1H, minor rotamer), 2.48 (m, 1H, majorrotamer), 2.21 (m, 1H, major rotamer), 2.07 (m, 1H, minor rotamer), 1.89(s, 3H)

¹³C-NMR (75 MHz; CD₃OD): (carbonyl and/or amidine carbons, mixture ofrotamers) δ 171.9, 171.2, 165.0, 162.8, 160.4

APCI-MS: (M+1)=503/505 m/z.

Preparation of Compound F(Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)-Aze-Pab×TFA)

(i) Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)-Aze-Pab(Teoc)

To a solution of Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)OH (940 mg, 3.78mmol; see Preparation C (v) above) in DMF (30 mL) under nitrogen at 0°C. was added HAze-Pab(Teoc).HCl (2.21 g, 4.91 mmol), PyBOP (2.16 g, 4.15mmol), and DIPEA (1.22 g, 9.45 mmol). The reaction was stirred at 0° C.for 2 h and then at room temperature for 4 h. The mixture wasconcentrated in vacuo and the residue chromatographed twice on silicagel, eluting first with CHCl₃:EtOH (15:1) and second with EtOAc:EtOH(20:1) to afford the sub-title compound (450 mg, 20%) as a crushablewhite foam.

Mp: 80–88° C.

R_(f)=0.60 (10:1 CHCl₃:EtOH) ¹

H NMR (300 MHz, CD₃OD, complex mixture of rotamers) δ 7.79 (d, J=8 Hz,2H), 7.42 (d, J=8 Hz, 2H), 7.05–7.08 (m, 1H), 6.93–6.99 (m, 2H),5.08–5.13 (m, 1H), 4.75–4.80 (m, 2H), 4.60–4.68 (m, 1H), 3.95–4.55 (m,8H), 2.10–2.75 (m, 2H), 1.05–1.11 (m, 2H), 0.08 (s, 9H).

APCI-MS: (M+1)=607 m/z.

(ii) Compound F

Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.357 g, 0.589 mmol;see step (i) above), was dissolved in 10 mL of TFA and allowed to reactfor 40 min. TFA was evaporated and the residue was freeze dried fromwater/acetonitrile to yield 0.33 g (93%) of the title compound as itsTFA salt.

¹H-NMR (600 MHz; CD₃OD) rotamers: δ 7.8–7.7 (m, 2H), 7.54 (d, 2H), 7.08(s, 1H, major rotamer), 7.04 (s, 1H, minor rotamer), 6.99 (s, 1H, majorrotamer), 6.95 (s, 1H), 6.92 (s, 1H, minor rotamer), 5.18 (m, 1H, minorrotamer), 5.14 (s, 1H, major rotamer), 5.08 (s, 1H, minor rotamer), 4.80(m, 1H, major rotamer), 4.73 (m, 1H), 4.65 (m, 1H), 4.6–4.4 (m, 2H),4.35 (m, 1H, major rotamer), 4.21 (doublet of multiplets, 2H), 4.12 (m,1H, major rotamer), 4.06 (m, 1H, minor rotamer), 3.99 (m, 1H, minorrotamer), 2.69 (m, 1H, minor rotamer), 2.53 (m, 1H, major rotamer), 2.29(m, 1H, major rotamer), 2.14 (m, 1H, minor rotamer).

¹³C-NMR (150 MHz; CD₃OD): (carbonyl and/or amidine carbons) δ 172.8,172.1, 167.4.

ESI-MS+: (M+1)=463 (m/z)

Preparation of Compound G (Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH))

(i) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH, Teoc)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.148 g, 0.24 mmol; seePreparation A step (ix) above), was dissolved in 9 mL of acetonitrileand 0.101 g (1.45 mmol) of hydroxylamine hydrochloride was added. Themixture was heated at 70° C. for 2.5 h, filtered through Celite® andevaporated. The crude product (0.145 g; 75% pure) was used directly inthe next step without further purification.

(ii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH, Teoc) (0.145 g, 0.23 mmol;see step (i) above), was dissolved in 0.5 mL of CH₂Cl₂ and 9 mL of TFA.The reaction was allowed to proceed for 60 minutes. TFA was evaporatedand the residue was purified using preparative HPLC. The fractions ofinterest were pooled and freeze-dried (2×), yielding 72 mg (yield overtwo steps 62%) of the title compound.

MS (m/z) 482 (M−1)⁻; 484 (M+1)⁺

¹H-NMR (400 MHz; CD₃OD): δ 7.58 (d, 2H), 7.33 (m, 3H), 7.15 (m, 2H),6.89 (t, 1H major rotamer), 6.86 (t, 1H minor rotamer), 5.18 (s, 1Hmajor rotamer; and m, 1H minor rotamer), 5.12 (s, 1H minor rotamer),4.77 (m, 1H major rotamer), 4.42 (m, 2H), 4.34 (m, 1H major rotamer),4.14 (m, 1H major rotamer), 4.06 (m, 1H minor rotamer), 3.95 (m, 1Hminor rotamer), 2.66 (m, 1H minor rotamer), 2.50 (m, 1H major rotamer),2.27 (m, 1H major rotamer), 2.14 (m, 1H minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidine carbons, rotamers) δ172.4, 172.3, 172.0, 171.4 152.3, 152.1

Preparation of Compound H:Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2.6-diF)(OH)

(i) Boc-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)

Boc-(S)Aze-OH (1.14 g, 5.6 mmol) was dissolved in 45 mL of DMF.4-Aminomethyl-2,6-difluorobenzonitrile (1.00 g, 5.95 mol, see Example1(xiv) above), PyBOP (3.10 g, 5.95 mmol) and DIPEA (3.95 mL, 22.7 mmol)were added and the solution was stirred at room temperature for 2 h. Thesolvent was evaporated and the residue was partitioned between H₂O andEtOAc (75 mL each). The aqueous phase was extracted with 2×50 mL EtOAcand the combined organic phase was washed with brine and dried overNa₂SO₄. Flash chromatography (SiO₂, EtOAc/heptane (3/1)) yielded thesub-title compound (1.52 g, 77%) as an oil which crystallized in therefrigerator.

¹H-NMR (400 MHz; CD₃OD): δ 7.19 (m, 2H), 4.65–4.5 (m, 3H), 3.86 (m, 1H),3.73 (m, 1H), 2.45–2.3 (m, 2H), 1.39 (s, 9H)

(ii) H-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)×HCl

Boc-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN) (0.707 g, 2.01 mmol, see step (i)above) was dissolved in 60 mL of EtOAc saturated with HCl(g). Afterstirring at room temperature for 15 minutes, the solvent was evaporated.The residue was dissolved in CH₃CN/H₂O (1/1) and was freeze-dried togive the sub-title compound (0.567 g, 98%) as an off-white amorphouspowder.

¹H-NMR (400 MHz; CD₃OD): δ 7.49 (m, 2H), 4.99 (m, 1H), 4.58 (m, 2H),4.12 (m, 1H), 3.94 (m, 1H), 2.80 (m, 1H), 2.47 (m, 1H)

MS (m/z) 252.0 (M+1)⁺

(iii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)

Ph(3-C1)(5-OCHF₂)—(R)CH(OH)C(O)OH (0.40 g, 1.42 mmol, see Example1(viii) above) was dissolved in 10 mL of DMF andH-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)×HCl (0.43 g, 1.50 mmol, see step (ii)above) and PyBOP (0.779 g, 1.50 mmol) were added, followed by DIPEA (1.0mL, 5.7 mmol). After stirring at room temperature for 2 h, the solventwas evaporated. The residue was partitioned between H₂O (200 mL) andEtOAc (75 mL). The aqueous phase was extracted with 2×75 mL EtOAc andthe combined organic phase was washed with brine and dried over Na₂SO₄.Flash chromatography (SiO₂, EtOAc/heptane (4/1)) yielded the sub-titlecompound (0.56 g, 81%) as an oil.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.43 (m, 2H), 7.31 (m, 1H, majorrotamer), 7.26 (m, 1H, minor rotamer), 7.2–7.1 (m, 2H), 6.90 (t, 1H,major rotamer), 6.86 (t, 1H, minor rotamer), 5.14 (s, 1H, majorrotamer), 5.11 (m, 1H, minor rotamer), 5.04 (s, 1H, minor rotamer), 4.71(m, 1H, major rotamer), 4.6–4.45 (m, 2H), 4.30 (m, 1H, major rotamer),4.2–3.9 (m, 1H; and 1H, minor rotamer), 2.62 (m, 1H, minor rotamer),2.48 (m, 1H, major rotamer), 2.21 (m, 1H, major rotamer), 2.09 (m, 1H,minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl carbons) δ 171.9, 171.8

MS (m/z) 484.0, 485.9 (M−1)⁻, 486.0, 487.9 (M+1)⁺

(iv) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN) (0.555 g,1.14 mmol, from step (iii) above) was dissolved in 10 mL of EtOH (95%).To this solution was added hydroxylamine hydrochloride (0.238 g, 3.42mmol) and Et₃N (0.48 mL, 3.44 mmol). After stirring at room temperaturefor 14 h, the solvent was removed and the residue was dissolved inEtOAc. The organic phase was washed with brine and H₂O and was driedover Na₂SO₄. The crude product was purified by preparative RPLC withCH₃CN:0.1 M NH4OAc as eluent, yielding the title compound as anamorphous powder (0.429 g, 72%) after freeze-drying.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.35–7.1 (m, 5H), 6.90 (t, 1H, majorrotamer), 6.85 (t, 1H, minor rotamer), 5.15 (s, 1H, major rotamer), 5.12(m, 1H, minor rotamer), 5.08 (s, 1H, minor rotamer), 4.72 (m, 1H, majorrotamer), 4.6–4.4 (m, 2H), 4.30 (m, 1H, major rotamer), 4.12 (m, 1H,major rotamer), 4.04 (m, 1H, minor rotamer), 3.94 (m, 1H, minorrotamer), 2.62 (m, 1H, minor rotamer), 2.48 (m, 1H, major rotamer), 2.22(m, 1H, major rotamer), 2.10 (m, 1H, minor rotamer)

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and amidine carbons, rotamers) δ172.4, 171.9, 171.0, 152.3, 151.5

MS (m/z) 517.1, 519.0 (M−1)⁻, 519.1, 521.0 (M+1)⁺

Preparation of Compound J(Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH))

(i) Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(Z)

Boc-Aze-Pab(Z) (see international patent application WO 97/02284, 92 mg,0.197 mmol) was dissolved in 10 mL of EtOAc saturated with HCl(g) andallowed to react for 10 min. The solvent was evaporated and the residuewas mixed with Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)OH (50 mg, 0.188 mmol;see Preparation C (v) above), PyBOP (109 mg, 0.209 mmol) and finallydiisopropylethyl amine (96 mg, 0.75 mmol) in 2 mL of DMF. The mixturewas stirred for 2 h and then poured into 50 mL of water and extractedthree times with EtOAc. The combined organic phase was washed withwater, dried (Na₂SO₄) and evaporated. The crude product was flashchromatographed on silica gel with EtOAc:MeOH (9:1). Yield: 100 mg(87%).

¹H NMR (300 MHz, CD₃OD, mixture of rotamers) δ 7.85–7.75 (m, 2H),7.45–7.25 (m, 7H), 7.11 (m, 1H, major rotamer), 7.08 (m, 1H, minorrotamer), 7.05–6.9 (m, 2H), 6.13 (bt, 1H), 5.25–5.05 (m, 3H), 4.77 (m,1H, partially hidden by the CD₃OH signal), 4.5–3.9 (m, 7H), 2.64 (m, 1H,minor rotamer), 2.47 (m, 1H, major rotamer), 2.25 (m, 1H, majorrotamer), 2.13 (m, 1H, minor rotamer)

(ii) Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(OH)

Hydroxylamine hydrochloride (65 mg, 0.94 mmol) and triethylamine (0.319g, 3.16 mmol) were mixed in 8 mL of THF and sonicated for 1 h at 40° C.Ph(3-Cl)(5-OCH₂CHF₂)—(R)CH(OH)C(O)-Aze-Pab(Z) (96 mg, 0.156 mmol; seestep (i) above) was added with 8 mL more of THF. The mixture was stirredat 40° C. for 4.5 days. The solvent was evaporated and the crude productwas purified by preparative RPLC with CH₃CN:0.1M NH₄OAc (40:60). Yield:30 mg (38%). Purity: 99%.

¹H NMR (300 MHz, CD₃OD mixture of rotamers) δ 7.6–7.55 (m, 2H), 7.35–7.3(m, 2H), 7.12 (m, 1H, major rotamer), 7.09 (m, 1H, minor rotamer),7.05–6.9 (m, 2H), 6.15 (triplet of multiplets, 1H), 5.15 (m, 1H, minorrotamer), 5.13 (s, 1H, major rotamer), 5.08 (s, 1H, minor rotamer), 4.77(m, 1H, major rotamer), 4.5–4.2 (m, 5H), 4.08 (m, 1H, major rotamer),3.97 (m, 1H, minor rotamer), 2.66 (m, 1H, minor rotamer), 2.50 (m, 1H,major rotamer), 2.27 (m, 1H, major rotamer), 2.14 (m, 1H, minorrotamer).

¹³C-NMR (100 MHz; CD₃OD): (carbonyl and/or amidine carbons, mixture ofrotamers) δ 172.8, 172.2, 171.4, 159.1, 158.9, 154.2.

APCI-MS: (M+1)=497/499 m/z

Methods 1 and 2 Preparation of Salts of Compound A Method 1 GeneralMethod for Salt Preparation

The following generic method was employed to prepare salts of CompoundA: 200 mg of Compound A (see Preparation A above) was dissolved in 5 mLof MeOH. To this solution was added a solution of the relevant acid (1.0molar equivalent) dissolved in 5 mL of MeOH. After stirring for 10minutes at room temperature, the solvent was removed by way of a rotaryevaporator. The remaining solid material was re-dissolved in 8 mL ofacetonitrile:H₂O (1:1). Freeze-drying afforded colorless amorphousmaterial in each case.

Acids Employed:

-   (1S)-(+)-10-camphorsulfonic-   malic-   cyclohexylsulphamic-   phosphoric-   dimethylphosphoric-   p-toluenesulphonic-   L-lysine-   L-lysine hydrochloride-   saccharinic-   methanesulphonic-   hydrochloric

Appropriate characterising data are shown in Table 1.

TABLE 1 δ ppm (MeOD) H18, H19, H24 (see structure Mw Mw at end of Saltacid salt LRMS Method 9 below) (1S)-(+)-10-camphor- 232.20 729.20 230.97.57, 7.68, 3.97 sulfonate 495.1 497.0 727.3 maleate 116.07 612.97 114.87.45, 7.64, 3.89 495.1 497.0 cyclohexylsulphamate 179.24 676.14 177.97.44, 7.64, 3.89 495.1 496.9 674.3 676.1 phosphate 97.99 594.89 495.17.37, 7.61, 3.84 497.0 593.1 dimethylphosphate 126.05 622.95 124.9 7.50,7.66, 3.92 495.1 497.0 621.2 623.0 p-toluenesulphonate 172.20 669.10170.9 7.54, 7.71, 3.95 495.1 497.0 L-lysine 146.19 643.09 145.0 7.36,7.60, 3.83 495.1 497.0 L-lysine hydrochloride 182.65 679.55 495.1 7.36,7.60, 3.83 497.0 531.1 (HC saccharinate 183.19 680.09 181.9 7.44, 7.64.3.89 495.1 497.0 methanesulphonate 96.11 593.01 495.1 7.57, 7.68, 3.97497.0 591.2 593.1 hydrochloride 36.46 533.36 495.1 7.55, 7.67, 3.95496.9 531.1 532.5 535.2All salts formed in this Method were amorphous.

Method 2

Further amorphous salts of Compound A were made using analogoustechniques to those described in Method 1 above from the followingacids:

-   hydrobromic acid (1:1 salt)-   hydrochloric acid (1:1 salt)-   sulphuric acid (1:0.5 salt)-   1,2-ethanedisulfonic acid (1:0.5 salt)-   1S-camphorsulfonic acid (1:1 salt)-   (+/−)-camphorsulfonic acid (1:1 salt)-   ethanesulfonic acid (1:1 salt)-   nitric acid (1:1 salt)-   toluenesulfonic acid (1:1 salt)-   methanesulfonic acid (1:1 salt)-   p-xylenesulfonic acid (1:1 salt)-   2-mesitylenesulfonic acid (1:1 salt)-   1,5-naphthalenesulfonic acid (1:0.5 salt)-   naphthalenesulfonic acid (1:1 salt)-   benzenesulfonic acid (1:1 salt)-   saccharinic acid (1:1 salt)-   maleic acid (1:1 salt)-   phosphoric acid (1:1 salt)-   D-glutamic acid (1:1 salt)-   L-glutamic acid (1:1 salt)-   D,L-glutamic acid (1:1 salt)-   L-arginine (1:1 salt)-   L-lysine (1:1 salt)-   L-lysine hydrochloride (1:1 salt)-   glycine(1:1 salt)-   salicylic acid (1:1 salt)-   tartaric acid (1:1 salt)-   fumaric acid (1:1 salt)-   citric acid (1:1 salt)-   L-(−)-malic acid (1:1 salt)-   D,L-malic acid (1:1 salt)-   D-gluconic acid (1:1 salt)

Method 3 Preparation of Amorphous Compound A, Ethanesulfonic Acid Salt

Compound A (203 mg; see Preparation A above) was dissolved in ethanol (3mL) and ethanesulfonic acid (1 eq., 95%, 35 μL) was added to thesolution. The mixture was stirred for a few minutes, and then thesolvent was evaporated. The resulting oil was slurried in iso-octane andevaporated to dryness until a solid material was obtained. Finally, thesubstance was re-slurried in iso-octane and the solvent evaporated againresulting in a white, dry, amorphous solid. The substance was vacuumdried at 40° C. overnight.

Methods 4 to 9 Preparation of Crystalline Compound A, EthanesulfonicAcid Salt Method 4 Crystallisation of Amorphous Material

Amorphous Compound A, ethanesulfonic acid salt (17.8 mg; see Method 3above) was slurried in methyl iso-butyl ketone (600 μL). After 1 week,crystalline needles were observed, which were filtered off andair-dried.

Methods 5 to 7 Reaction Crystallisations (without Anti-Solvent) Method 5

Compound A (277 mg; see Preparation A above) was dissolved in methyliso-butyl ketone (3.1 mL). Ethanesulfonic acid was added (1 eq., 95%, 48μL). Precipitation of amorphous ethanesulfonate salt occurredimmediately. More methyl iso-butyl ketone (6 mL) was added and theslurry was treated with ultrasound. Finally, a third portion of methyliso-butyl ketone (3.6 mL) was added and then the slurry was leftovernight with stirring (magnetic stirrer). The next day, the substancehad transformed into crystalline needles. The slurry was filtered off,washed with methyl iso-butyl ketone (0.5 mL) and air dried.

Method 6

Compound A (236 mg; see Preparation A above) was dissolved at roomtemperature in methyl iso-butyl ketone (7 mL). Ethanesulfonic acid (1eq., 41 μL) was mixed with 2 mL of methyl iso-butyl ketone in a vial.The solution of Compound A was seeded with crystalline Compound A,ethanesulfonic acid salt (see Methods 4 and 5 above). Then, 250 μL ofthe methyl iso-butyl ketone solution of ethanesulfonic acid was added inportions over 45 minutes. The solution was seeded again, and thetemperature was increased to 30° C. Then, 500 μL of the methyl iso-butylketone solution was added over approximately 1 hour. The resultingslurry was left overnight before a final amount of the methyl iso-butylketone/acid solution was added over 20 minutes. The vial was rinsed with1.5 mL of methyl iso-butyl ketone, which was added to the slurry. Aftera further 6 hours, the crystals were filtered off, washed with methyliso-butyl ketone (2 mL) and dried under reduced pressure at 40° C. Atotal of 258 mg of crystalline salt was obtained which corresponds to ayield of approximately 87%.

Method 7

Compound A (2.36 g; see Preparation A above) was dissolved in methyliso-butyl ketone (90 mL). Seed crystals (10 mg) of Compound A,ethanesulfonic acid salt (see Methods 4 to 6 above) were added to thesolution, and then ethanesulfonic acid (40 TL) was added in twoportions. Further seed crystals (12 mg) and two portions ofethanesulfonic acid (2×20 μL) were then added. The slurry was dilutedwith methyl iso-butyl ketone (15 mL) before the addition ofethanesulfonic acid was continued. A total amount of 330 μLethanesulfonic acid was added, in portions, over 1 hour. A small amountof seed crystals was added and, finally, the slurry was left overnightwith stirring. The next day, the crystals were filtered off, washed withmethyl iso-butyl ketone (2×6 mL) and dried under reduced pressure at 40°C. After drying, a total of 2.57 g of white, crystalline product wasobtained corresponding to a yield of 89%.

Methods 8 and 9 Reaction Crystallizations (with Anti-Solvent) Method 8

Compound A (163 mg; see Preparation A above) was dissolved iniso-propanol (1.2 mL). The solution was heated to 35° C. Ethanesulfonicacid was added (28 μL). Then, ethyl acetate (4.8 mL) was added and thesolution was seeded with crystalline Compound A, ethanesulphonic acidsalt (see Methods 4 to 7 above). Crystallization started almostimmediately. The slurry was left for about 80 minutes at 35° C. beforebeing allowed to cool to ambient temperature (21° C.). Two hours later,the crystals were filtered off, washed three times with ethyl acetate(3×0.4 mL), and dried under reduced pressure at 40° C. A total of 170 mgof crystalline title product was obtained which corresponds to a yieldof approximately 82%.

Method 9

Compound A (20.0 g; see Preparation A above) was dissolved iniso-propanol (146.6 mL) at 40° C. and ethanesulfonic acid (3.46 mL, 95%,1 eq.) was added to the solution. To the resulting clear solution, seedcrystals of Compound A, ethanesulfonic acid salt were added (50 mg; seeMethods 4 to 8 above). Then, ethyl acetate (234 mL) was added over 10minutes. The resulting slightly opaque solution was seeded once more (70mg) and left for one hour at 40° C. with stirring to allow forcrystallization to start. After this, a total of 352 mL of ethyl acetatewas added at a constant rate over one hour. When all of the ethylacetate had been added, the slurry was left for 1 hour, before beingcooled to 21° C. over 2 hours. The crystallization was allowed tocontinue for 1 hour at 21° C. before the crystals were filtered off,washed twice with ethyl acetate (50 mL+60 mL) and finally, dried underreduced pressure at 40° C. overnight. A total of 21.6 g of a white,crystalline salt was obtained, corresponding to a yield of approximately90%.

Compound A, ethanesulfonic acid salt was characterised by NMR asfollows: 23 mg of the salt was dissolved in deuterated methanol (0.7 mL)troscopy. A combination of ID (¹H, ¹³C and selective NOE) and 2D (gCOSY,gHSQC and gHMBC) NMR experiments were used. All data were in goodagreement with the theoretical structure of the salt, shown below. Themolecule exists in two conformations in methanol. Based on the integralof the peak assigned to H5 (dominant conformer) and peak assigned to H5′(other conformer), the ratio between the two conformers was found to be70:30. H22 could not be observed as these protons were in fast exchangewith the solvent CD₃OD.

Both the proton and the carbon resonance corresponding to position 1 aresplit due to the spin-coupling with the two fluorine nuclei in thatposition. The coupling constants are ²J_(HF)=73 Hz and ¹J_(CF)=263 Hz.

¹H and 13C NMR chemical shift assignment and proton-proton correlationsare shown in Table 2.

TABLE 2 Atom ¹³C shift/ ¹H shift/ppm^(b) and No. Type ppm^(a)multiplicity^(c) J_(HH)/Hz  1 CH 117.5^(e) 6.90 (t) 73 (²J_(HF))  1′117.5^(e) 6.88 (t)  2 C 153.5  2′ 153.5  3 CH 120.0 7.15 (s)  3′ 119.77.13 (s)  4 C 136.2  4′ 135.9  5 CH 125.0 7.36 (s)  5′ 124.9 7.31 (s)  6C 144.5  6′ 145.3  7 CH 117.3 7.20 (s)  7′ 117.2 7.15 (s)  8 CH  72.05.20 (s)  8′  74.0 5.12 (s)  9 CO 173.1  9′ 173.8  11 CH₂  51.6 a: 4.38(m) b: 4.21 (m)  11′  49.0 a: 4.06 (m) b: 3.99 (m)  12 CH₂  21.7 a: 2.55(m) b: 2.29 (m)  12′  23.2 a: 2.70 (m) b: 2.15 (m)  13 CH  63.1 4.80 (m) 13′  66.2 5.22 (m)  14 CO 172.9  14′ 173.6  15 NH 8.76 (t, br)  5.2 15′ 8.79 (t, br)  5.2  16 CH₂  43.5 4.59 (AB-pattern) 15.9 4.46(AB-pattern) 15.9  16′  43.6 4.53 (AB-pattern) 15.9 4.49 (AB-pattern)15.9  17 C 146.9  17′ 147.0  18 CH 129.1 7.56 (d)  7.8  18′ 129.1 7.57(d)  7.8  19 CH 129.2 7.67 (d)  7.8  19′ 129.4 7.70 (d)  7.8  20 C 124.9—  20′ 124.9  21 C 162.4  21′ 162.3  22 NH₂ Not observed  24 CH₃  64.83.96 (s) 101 CH3 1.28 (t)  7.4 102 CH2 2.77 (m)  7.4 ^(a)Relative to thesolvent resonance at 49.0 ppm. ^(b)Relative to the solvent resonance at3.30 ppm. ^(c)s = singlet, t = triplet, m = multiplet, br = broad, d =doublet ^(d)Obtained in the gCOSY experiment. ^(e)The resonance is atriplet due to coupling with the two fluorine nuclei. ¹J_(CF) = 263 Hz.

HRMS calculated for C₂₄H₂₉ClF₂N₄O₈S (M−H)⁻ 605.1284, found 605.1296.

Crystals of Compound A, ethanesulfonic acid salt (obtained by way of oneor more of Examples 4 to 9 above) were analyzed by XRPD and the resultsare tabulated below (Table 3) and are shown in FIG. 1.

TABLE 3 d value (Å) Intensity (%) Intensity 16.5 10 m 12.2 74 vs 11.0 4w 9.0 33 s 8.3 3 vw 7.6 6 w 6.4 4 w 6.2 12 m 6.0 7 m 5.9 10 m 5.5 15 m5.4 100 vs 5.1 7 m 4.66 29 s 4.60 36 s 4.31 57 s 4.25 18 m 4.19 20 m4.13 12 m 4.00 12 m 3.87 13 m 3.83 6 w 3.76 7 m 3.72 6 w 3.57 9 m 3.51 7m 3.47 5 w 3.39 3 vw 3.31 11 m 3.26 10 m 3.21 8 m 3.16 4 w 3.03 8 m 2.784 w 2.74 5 w 2.67 3 vw 2.56 5 w 2.50 5 w 2.46 7 m 2.34 4 w 2.21 5 w 2.003 vw 1.98 3 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca. 131° C. TGA showed a decrease in mass of ca. 0.2% (w/w) aroundthe melting point. DSC analysis repeated with a sample of lower solventcontent showed a melting onset temperature of ca. 144° C.

Method 10 Preparation of Amorphous Compound A, Benzenesulfonic Acid Salt

Compound A (199 mg; see Preparation A above) was dissolved in ethanol (2mL). Benzenesulfonic acid (1 eq. 90%, 70 mg) was dissolved in ethanol (1mL) in a vial. The ethanol solution of the acid was added to thesolution of Compound A and the vial was rinsed with 1 mL ethanol, whichwas then added to the mixture. The mixture was stirred for a fewminutes, and then the ethanol was evaporated until an oil was formed.Ethyl acetate (3 mL) was added and the solvent was evaporated again todryness. An amorphous solid was formed.

Methods 11 to 13 Preparation of Crystalline Compound A, BenzenesulfonicAcid Salt Method 11 Crystallisation of Amorphous Material

Amorphous Compound A benzenesulfonic acid salt (20.7 mg; see Method 10above) was slurried in ethyl acetate (600 TL). After 5 days, crystallineneedles were observed in the slurry.

Methods 12 and 13 Reaction Crystallisations Method 12

Compound A (128 mg; see Preparation A above) was dissolved in ethylacetate (3 mL). The solution was seeded with the slurry from Method 11above. Then, benzenesulfonic acid was added (1 eq., 90%, 45 mg).Precipitation of benzenesulphonic acid salt occurred immediately.iso-Propanol was added to the slurry (0.8 mL) and the mixture was seededagain. Two days later, the substance had transformed into crystallineneedles. The slurry was filtered off, washed with ethyl acetate (3×0.2mL) and dried for a short time under vacuum at 40° C. A total ofapproximately 140 mg of white solid was obtained.

Method 13

Compound A (246 mg; see Preparation A above) was dissolved iniso-propanol (1.52 mL). Benzenesulfonic acid was added (88 mg, 90%). Tothe clear solution, ethyl acetate was added (3 mL), and then the mixturewas seeded to initiate crystallisation. After 1 hour, more ethyl acetatewas added (2.77 mL). Finally, the slurry was allowed to crystalliseovernight before the crystals were filtered off, washed with ethylacetate (3×0.3 mL) and dried at 40° C. under vacuum. A total of 279 mgsalt was obtained which corresponds to a yield of approximately 86%.

Compound A, benzenesulfonic acid salt was characterised by NMR asfollows: 20 mg of the salt was dissolved in deuterated methanol (0.7mL). A combination of 1D (¹H, ¹³C and selective NOE) and 2D (gCOSY,gHSQC and gHMBC) NMR experiments were used. All data were in goodagreement with the theoretical structure of the salt, shown below. Themolecule exists in two conformations in methanol. Based on the integralof the peak assigned to H12 (dominant conformer) and peak assigned toH12′ (other conformer), the ratio between the two conformers was foundto be 70:30. H22 could not be observed as these protons were in fastexchange with the solvent CD₃OD.

Both the proton and the carbon resonance corresponding to position 1 aresplit due to the spin-coupling with the two fluorine nuclei in thatposition. The coupling constants are ²J_(HF)=74 Hz and ¹J_(CF)=260 Hz.

¹H and ¹³C NMR chemical shift assignment and proton-proton correlationsare shown in Table 4.

TABLE 4 Atom ¹H shift/ppm^(b) and No. Type ¹³C shift/ppm^(a)multiplicity^(c) J_(HH)/Hz  1 CH 117.5^(e) 6.89 (t) 74 (²J_(HF))  1′117.5^(e) 6.87 (t)  2 C 153.5  2′ 153.5  3 CH 120.1 7.15 (s)  3′ 119.77.12 (s)  4 C 136.2  4′ 135.9  5 CH 125.1 7.35 (s)  5′ 124.9 7.31 (s)  6C 144.5  6′ 145.3  7 CH 117.3 7.20 (s)  7′ 117.2 7.14 (s)  8 CH  72.85.20 (s)  8′  74.0 5.12 (s)  9 CO 173.1  9′ 173.8  11 CH₂  51.6 a: 4.37(m) b: 4.20 (m)  11′  49.0 a: 4.05 (m) b: 3.98 (m)  12 CH₂  21.7 a: 2.53(m) b: 2.28 (m)  12′  23.2 a: 2.69 (m) b: 2.14 (m)  13 CH  63.1 4.79 (m) 13′  66.2 5.22 (m)  14 CO 172.9  14′ 173.6  15 NH 8.75 (t, br)  5.3 15′ 8.78 (t, br)  5.3  16 CH₂  43.5 4.59 (AB-pattern) 16.0 and 5.2 4.44(AB-pattern) 16.0 and 4.8  16′  43.6 4.51 (AB-pattern) 16.0 4.46(AB-pattern) 16.0  17 C 146.9  17′ 147.0  18 CH 129.2 7.54 (d)  8.3  18′129.2 7.56 (d)  8.3  19 CH 129.3 7.66 (d)  8.3  19′ 129.4 7.69 (d)  8.3 20 C 124.9 —  20′ 124.9  21 C 162.4  21′ 162.4  22 NH₂ Not observed  24CH₃  64.8 3.95 (s) 101 CH 126.9 7.81 (m) 102 CH 129.1 7.41 (m) 103 CH131.2 7.42 (m) 104 C 146.4 ^(a)Relative to the solvent resonance at 49.0ppm. ^(b)Relative to the solvent resonance at 3.30 ppm. ^(c)s = singlet,t = triplet, m = multiplet, br = broad, d = doublet. ^(d)Obtained in thegCOSY experiment. ^(e)The resonance is a triplet due to coupling withthe two fluorine nuclei. ¹J_(CF) = 260 Hz. ^(f)connectivity difficult todetermine due to overlap between resonance 102 and 103

HRMS calculated for C₂₈H₂₉ClF₂N₄O₈S (M−H)⁻ 653.1284, found 653.1312.

Crystals of Compound A, benzenesulfonic acid salt (obtained by way ofone or more of Examples 11 to 13 above) were analyzed by XRPD and theresults are tabulated below (Table 5) and are shown in FIG. 2.

TABLE 5 d value (Å) Intensity (%) Intensity 14.2 12 m 12.6 55 s 10.2 49s 7.5 8 m 6.4 5 w 6.3 30 s 6.1 5 w 5.9 100 vs 5.7 20 m 5.4 9 m 5.3 11 m5.1 10 m 4.96 3 vw 4.83 27 s 4.73 72 vs 4.54 23 s 4.50 10 m 4.35 28 s4.30 38 s 4.24 24 s 4.17 28 s 4.09 60 vs 4.08 61 vs 3.96 29 s 3.91 15 m3.77 22 s 3.62 11 m 3.52 20 m 3.31 44 s 3.19 8 m 3.15 11 m 3.09 8 m 3.007 m 2.89 3 vw 2.86 4 w 2.79 7 m 2.76 6 w 2.72 5 w 2.59 6 w 2.56 9 m 2.549 m 2.49 7 m 2.38 8 m 2.16 4 w 2.03 3 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca. 152° C. TGA showed a decrease in mass of ca. 0.1% (w/w) aroundthe melting point.

Method 14 Preparation of Amorphous Compound A, n-propanesulfonic AcidSalt

Compound A (186 mg; see Preparation A above) was dissolved iniso-propanol (1.39 mL) and n-propanesulfonic acid (1 eq., 95%, 39 TL)was added. Ethyl acetate (5.6 mL) was added and the solvent wasevaporated until a dry, amorphous solid was formed.

Methods 15 and 16 Preparation of Crystalline Compound A,n-propanesulfonic Acid Salt Method 15 Crystallisation of AmorphousMaterial

Amorphous Compound A, n-propanesulfonic acid salt (20 mg; see Method 14above) was dissolved in iso-propanol (60 TL) and iso-propyl acetate (180TL) was added. After three days crystalline needles were observed.

Method 16 Reaction Crystallisation

Compound A (229 mg; see Preparation A above) was dissolved iniso-propanol (1.43 mL). n-Propanesulfonic acid was added (1 eq., 95%, 48TL). Ethyl acetate was added (2 mL), and then the solution was seededwith crystalline salt from Method 15 above. Further ethyl acetate wasadded (5 mL) and the slurry was left overnight to crystallize. Thecrystals were filtered off, washed with ethyl acetate (3×0.3 mL) anddried under vacuum at 40° C.

Compound A, n-propanesulfonic acid salt was characterised by NMR asfollows: 13 mg of the salt was dissolved in deuterated methanol (0.7 mL)troscopy. A combination of 1D (¹H, 13C) and 2D (gCOSY) NMR experimentswere used. All data were in good agreement with the theoreticalstructure of the salt, shown below. The molecule exists in twoconformations in methanol. Based on the integral of the peak assigned toH12 (dominant conformer) and peak assigned to H12′ (other conformer),the ratio between the two conformers was found to be 65:35. H22 couldnot be observed as these protons were in fast exchange with the solventCD₃OD.

Both the proton and the carbon resonance corresponding to position 1 aresplit due to the spin-coupling with the two fluorine nuclei in thatposition. The coupling constants are ²J_(HF)=74 Hz and ¹J_(CF)=260 Hz.

¹H and ¹³C NMR chemical shift assignment and proton-proton correlationsare shown in Table 6.

TABLE 6 Atom ¹H shift/ppm^(b) and No. Type ¹³C shift/ppm^(a)multiplicity^(c) J_(HH)/Hz  1 CH 117.5^(e) 6.89 (t) 74 (²J_(HF))  1′117.5^(e) 6.88 (t)  2 C 153.5  2′ 153.5  3 CH 120.0 7.16 (s)  3′ 119.77.13 (s)  4 C 136.2  4′ 135.9  5 CH 125.1 7.36 (s)  5′ 124.9 7.31 (s)  6C 144.5  6′ 145.3  7 CH 117.3 7.20 (s)  7′ 117.2 7.16 (s)  8 CH  72.95.20 (s)  8′  74.1 5.12 (s)  9 CO 173.1  9′ 173.8  11 CH₂  51.6 a: 4.37(m) b: 4.20 (m)  11′  49.0 a: 4.06 (m) b: 3.98 (m)  12 CH₂  21.7 a: 2.53(m) b: 2.29 (m)  12′  23.2 a: 2.69 (m) b: 2.15 (m)  13 CH  63.1 4.80 (m) 13′  66.2 5.22 (m)  14 CO 172.9  14′ 173.8  15 NH 8.75 (t, br)  5.5 15′ 8.79 (t, br)  5.5  16 CH₂  43.5 4.59 (AB-pattern) 16.0 and 6.6 4.45(AB-pattern) 16.0 and 5.3  16′  43.6 4.51 4.50  17 C 146.9  17′ 147.0 18 CH 129.1 7.54 (d)  8.5  18′ 129.2 7.57 (d)  8.5  19 CH 129.2 7.67(d)  8.5  19′ 129.4 7.69 (d)  8.5  20 C 124.9 —  20′ 124.9  21 C 162.4 21′ 162.4  22 NH₂ Not observed  24 CH₃  64.7 3.96 (s) 101 CH  13.7 1.0(t) 102 CH  19.6 1.78 (m) 103 CH  54.6 2.75 (m) ^(a)Relative to thesolvent resonance at 49.0 ppm. ^(b)Relative to the solvent resonance at3.30 ppm. ^(c)s = singlet, t = triplet, m = multiplet, br = broad, d =doublet. ^(d)Obtained in the gCOSY experiment. ^(e)The resonance is atriplet due to coupling with the two fluorine nuclei. ¹J_(CF) = 260 Hz.

HRMS calculated for C₂₅H₃₁ClF₂N₄O₈S (M−H)⁻ 619.1441, found 619.1436.

Crystals of Compound A, n-propanesulfonic acid salt (obtained by way ofone or more of Examples 15 and 16 above) were analyzed by XRPD and theresults are tabulated below (Table 7) and are shown in FIG. 3.

TABLE 7 d value (Å) Intensity (%) Intensity 14.0 4 w 12.4 87 vs 10.0 30s 8.0 3 vw 7.5 7 m 7.0 0.6 vw 6.7 1 vw 6.4 1 vw 6.2 12 m 6.1 3 vw 5.8100 vs 5.7 11 m 5.5 3 vw 5.4 5 w 5.3 5 w 5.2 2 vw 5.1 3 vw 4.94 3 vw4.78 21 s 4.68 42 s 4.51 10 m 4.49 7 m 4.40 5 w 4.32 10 m 4.29 10 m 4.2522 s 4.19 14 m 4.14 15 m 4.07 23 s 4.04 20 m 3.94 16 m 3.88 10 m 3.73 15m 3.65 2 vw 3.59 3 vw 3.48 18 m 3.28 23 m 3.12 4 w 3.06 3 vw 2.97 6 w2.84 2 vw 2.81 3 vw 2.76 2 vw 2.73 3 vw 2.70 2 vw 2.57 2 vw 2.54 6 w2.51 6 w 2.46 8 m 2.42 2 vw 2.39 3 vw 2.36 3 vw 2.32 2 vw 2.14 3 vw 2.012 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca. 135° C. TGA showed no decrease in mass around the melting point.

Method 17 Method 17-A Preparation of Amorphous Compound A n-butaneSulfonic Acid Salt

Amorphous Compound A (277 mg) was dissolved in IPA (1.77 ml) and butanesulfonic acid (approx. 1 eq. 70 μL) was added. Ethyl acetate (6 ml) wasadded and the solvent was evaporated until dry, amorphous solid wasformed.

Method 17-B Preparation of Crystalline Compound A Butane Sulfonic AcidSalt

Amorphous Compound A butane sulfonic acid salt (71.5 mg; see preparationabove) was slurried in ethyl acetate (500 μl) over night. The crystalswere filtered off and were air-dried.

Compound A, butanesulfonic acid salt was charaterised by NMR as follows:21.6 mg of the salt was dissolved in deuterated dimethylsulfoxide (0.7ml) and was investigated with ¹H and ¹³C NMR spectroscopy.

The spectra are very similar to other salts of the same compound and ingood agreement with the structure shown below. Most resonances in thespectra are present as sets of two peaks due to the slow rotation aroundthe C9-N10 bond, which results in two atropisomers that simultaneouslyexist in the solution. This is shown for other salts of the samecompound.

The two fluorine nuclei in position 1 give rise to split resonances forthe proton and the carbon in that position. The coupling constants are²J_(HF)=73 Hz and ¹J_(CF)=258 Hz.

Chemical shifts for protons and carbons are presented in Table 1.Protons in position 22 and 24 are not detected due to chemical exchange.There is a very broad hump between 8 and 9 ppm in the proton spectrumcorresponding to these protons.

TABLE 8 ¹H and ¹³C NMR chemical shift assignment of Compound An-butanesulfonate salt in deuterated dimethylsulfoxide at 25° C. Atom¹³C shift/ ¹H shift/ppm^(b) and No. Type ppm^(a) multiplicity^(c)J_(HH)/Hz  1 CHF₂ 116.3^(d) 7.29 (t) 73 (²J_(HF))  1′ 116.3^(d) 7.28 (t)73 (²J_(HF))  2 C 151.5 na na  2′ 151.3 na na  3 CH 118.0 7.25 (t)^(e)nd  3′ 117.6 7.21 (t)^(e) nd  4 C 133.8 na na  4′ 133.4 na na  5 CH123.8 7.34 (t)^(e) nd  5′ 123.6 7.25 (t)^(e) nd  6 C 144.5 na na  6′145.2 na na  7 CH 116.3 7.19 (t)^(e) nd  7′ 116.1 7.12 (t)^(e) nd  8 CH 70.9 5.13 (s) na  8′  71.2 4.99 (s) na  9 CO 170.6 na na  9′ 171.1 nana 11 CH₂  50.0 a: 4.24 (m) b: 4.12 (m) nd 11′  46.9 3.85 (m) nd 12 CH₂ 20.5 a: 2.41 (m) b: 2.10 (m) nd 12′  21.7 a: 2.60 (m) b: 2.02 (m) nd 13CH  61.2 4.65 (dd)  5.6 and 8.9 13′  63.9 5.12 (m) nd 14 CO 170.2 na na14′ 171.0 na na 16 CH₂  41.8 4.38 (m) nd 16′  42.0 4.38 (m) nd 17 C144.7 na na 18 CH 127.5 7.44 (d)  8.2 127.6 7.44 nd 19 CH 127.8 7.66 (d) 8.2 20 C 125.1 na na 21 C 157.9 na na 24 CH₃  63.3 3.83 (s) na 24′ 63.3 3.82 (s) na 26 CH₂  51.4 2.41 (m) nd 27 CH₂  27.3 1.52 (m) nd 28CH₂  21.7 1.30 (m) nd 29 CH₃  14.0 0.83 (t)  7.3 ^(a)Relative to thesolvent resonance at 49.0 ppm. ^(b)Relative to the solvent resonance at3.30 ppm. ^(c)s = singlet, d = doublet, dd = doublet of doublets, t =triplet, m = multiplet. ^(d)The resonance is a triplet due to couplingwith the two fluorine nuclei F1. ¹J_(CF) = 258 Hz. ^(e)The ⁴J_(HH)coupling with the meta-protons is not fully resolved. na = notapplicable, nd = not determined

HRMS calculated for C₂₆H₃₂ClF₂N₄O₈S (M−H)⁻ 633.1597, found 633.1600

Crystals of Compound A n-butanesulfonic acid salt (obtained as describedabove in Method 17-B) were analyzed by XRPD and the results aretabulated below (Table 9) and are shown in FIG. 4.

TABLE 9 d-value (Å) Intensity (%) Intensity 14.3 8 m 12.8 81 vs 10.3 44s 8.2 4 w 7.7 13 m 6.7 2 vw 6.4 8 m 6.2 18 m 6.0 100 vs 5.8 29 s 5.6 4 w5.4 11 m 5.3 16 m 5.1 15 m 4.98 6.5 w 4.91 34 s 4.76 56 s 4.57 20 m 4.4213 m 4.36 19 m 4.30 45 s 4.18 42 s 4.13 88 vs 4.01 34 s 3.92 28 s 3.8218 m 3.64 6.6 w 3.58 16 m 3.47 5 w 3.44 6 w 3.38 12 m 3.35 32 s 3.32 22s 3.29 12 m 3.20 8 m 3.17 9 m 3.02 12 m 2.90 6 w 2.81 3.9 vw 2.75 3 vw2.64 3.5 vw 2.59 10 m 2.57 8 m 2.50 4 w 2.45 5 w 2.40 6 w 2.31 3 vw

DSC showed an endotherm with an extrapolated melting onset temperatureof ca 118° C. and TGA showed a 0.04% weight loss.

Method 18 Preparation of Salts of Compound B Method 18-A General Methodfor Salt Preparation

The following generic method was employed to prepare salts of CompoundB: 200 mg of compound B (see Preparation B above) was dissolved in 5 mLof MIBK (methyl isobutyl ketone). To this solution was added a solutionof the relevant acid (1.0 or 0.5 molar equivalent, as indicated in Table10) dissolved in 1.0 mL of MIBK. After stirring for 10 minutes at roomtemperature, the solvent was removed by way of a rotary evaporator. Theremaining solid material was re-dissolved in about 8 mL ofacetonitrile:H₂O (1:1). Freeze-drying afforded colorless amorphousmaterial in each case.

Acid Employed:

-   Esylate (ethanesulfonic acid)-   Besylate (benzene sulfonic acid)-   Cyclohexylsulphamate-   Sulphate-   Bromide-   p-Toluenesulphonate-   2-Naphtalenesulfonate-   Hemisulfate-   Methanesulphonate-   Nitrate-   Hydrochloride

Appropriate characterising data are shown in Table 10

TABLE 10 Salt Mw acid Mw salt MS ES- Esylate 110.13 643.01 108.8 531.1641.0 Besylate 158.18 691.06 156.8 531.1 689.2 Cyclohexyl- 179.24 712.12177.9 sulphamate 531.2 710.4 Sulphate 98.08 630.96 531.1 Bromide 80.91613.79 531.2 613.1 p-Toluenesulphonate 172.20 705.08 170.9 531.1 703.12- 208.24 741.12 206.9 Naphtalenesulfonate 531.1 739.3 Hemisulfate 98.071163.8 531.1 (1:2) 631.0 630.85 (1:1) Methanesulphonate 96.11 628.99531.1 627.1 Nitrate 63.01 595.89 531.0 594.0 Hydrochloride 36.46 569.34531.0 569.0

All salts formed in this Example were amorphous.

Method 18-B

Further amorphous salts of Compound B were made using analogoustechniques to those described in Method 18-A above for the followingacids:

-   1,2-Ethanedisulfonic (0.5 salt)-   1S-Camphorsulfonic-   (+/−)-Camphorsulfonic-   p-Xylenesulfonic-   2-Mesitylenesulfonic-   Saccharin-   Maleic-   Phosphoric-   D-glutamic-   L-arginine-   L-lysine-   L-lysine*HCl

Method 18-C Preparation of Amorphous Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Amorphous Compound B (110.9 mg) was dissolved in 2.5 mL 2-propanol and0.5 equivalent of 1,5-naphthalene-disulfonic acid tetrahydrate was added(dissolved in 1 mL 2-propanol). The sample was stirred overnight. Onlysmall particles (amorphous) or oil drops were observed by microscopy.The sample was evaporated to dryness.

Method 18-D Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

The crystallization experiment was carried out at ambient temperature.Amorphous Compound B (0.4 gram) was dissolved in ethanol (1.5 mL) and0.5 eq of 1,5-naphthalene-disulfonic acid tetrahydrate (1.35 gram, 10%in ethanol) was added. Heptane (0.7 mL) was then added until thesolution became slightly cloudy. After about 15 minutes the solutionbecame turbid. After about 30 minutes thin slurry was obtained andadditional heptane (1.3 mL) was added. The slurry was than leftovernight for ripening. To dilute the thick slurry, a mixture of ethanoland heptane (1.5 mL and 1.0 mL respectively) was added. After about 1hour the slurry was filtered and the crystals were washed with a mixtureof ethanol and heptane (1.5:1) and finally with pure heptane. Thecrystals were dried at ambient temperature in 1 day. The dry crystalsweighed 0.395 g.

Method 18-E Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Amorphous Compound B (1.009 gr) was dissolved in 20 mL 2-propanol+20 mLethyl acetate. 351.7 mg 1,5-naphtalene-disulfonic acid tetrahydrate,dissolved in 20 mL 2-propanol, was added drop by drop. Precipitationoccurred in about 5 minutes. The slurry was stirred over night and thenfiltered.

Method 18-F Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

430.7 mg of the 1,5-naphtalene-disulfonic acid salt was dissolved in 30mL 1-propanol. The solution was heated to boiling in order to dissolvethe substance. The solution was left over night at ambient temperaturefor crystallization and then the crystals were filtered off.

Method 18-G Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

The mother liquid from Method 18-F was evaporated and the solid rest(61.2 mg) was dissolved in 6 mL acetonitrile/1-propanol, ratio 2:1. Thesolution was left overnight at ambient temperature to crystallize andthen the crystals were filtered off.

Method 18-H Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

The sample from Method 18-C was dissolved in about 2 mL methanol.Ethanol (about 3 mL) was added as anti-solvent at ambient temperatureand seeds were added. No crystallization occurred, so solvents wereevaporated (about half of the amount) and a new portion of ethanol(about 2 mL) and seeds were added. Crystalline particles were formedwhen stirred at ambient temperature during night.

Method 18-I Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Amorphous Compound B (104.1 mg) was dissolved in 2-propanol and 1equivalent of 1,5-naphthalene-disulfonic acid tetrahydrate, dissolved in2-propanol, was added In total, the 2-propanol amount was about 2.5 mL.The solution was stirred at 44° C. for about 80 minutes and aprecipitate was formed. The particles were crystalline according topolarised light microscopy. The sample was filtered.

Method 18-J Preparation of Crystalline Compound B,hemi-1,5-naphtalenedisulfonic acid salt

Compound B, hemi-1,5-naphtalenedisulfonic acid salt (56.4 mg) wasdissolved in 1.5 mL methanol. Methyl ethyl ketone (3 mL) was added.Seeds were added to the solution and crystallization started. Thecrystals were filtered off, washed with methyl ethyl ketone and airdried.

Method 18-K Preparation of Crystalline Compound B,.hemi-1,5-naphtalenedisulfonic Acid Salt

Amorphous Compound B (161,0 mg) was dissolved in 3.5 mL 1-Butanol andthe solution was heated to 40° C. In another beaker 57.4 mg ofnaphthalene-disulfonic acid tetrahydrate was dissolved in 3 mL1-Butanol. A couple of drops of the acid solution were added to thesolution of compound B. Then seeds were added to the solution and after2 hours the rest of the acid solution was added (at 40° C.) slowly. Thenthe temperature was slowly decreased to room temperature and theexperiment was left under stirring overnight. The slurry was filtered,washed with 1-Butanol and dried under vacuum at 44° C. for 2 hours. Theyield was 83%.

Characterisation

Crystals of Compound B, hemi-1,5-naphtalenedisulfonic acid salt,obtained by way of Method 18-D above, was charaterised by NMR asfollows:

21.3 mg of the salt was dissolved in deuterated methanol, 0.7 ml wasinvestigated with NMR spectroscopy. A combination of 1D (¹H, 13C andselective NOE) and 2D (gCOSY, gHSQC and gHMBC) NMR experiments was used.

All data are in good agreement with the proposed structure, shown below.All carbons and the protons attached to carbons are assigned. Protonsattached to heteroatoms are exchanged for deuterium from the solvent andare not detected. Most resonances in the 1D ¹H and ¹³C NMR spectra arepresent as sets of two peaks. The reason for this is a slow rotationaround the C9-N10 bond, which results in two atropisomers thatsimultaneously exist in the solution. The 1D NOE experiment is anevidence for this. When a resonance of one atropisomer is irradiated,the saturation is transferred to the corresponding peak of the otheratropisomer. The resonances corresponding to the1,5-naphtalenedisulfonate counter ion do not show atropisomerism.

There are four fluorine atoms in the molecule. They give rise to splitresonances for some protons and carbons. Both the proton and the carbonresonance corresponding to position 1 are split due to the spincouplingwith the two fluorine nuclei in that position. The coupling constantsare ²J_(HF)=73 Hz and ¹J_(CF)=263 Hz. Further, the proton resonancecorresponding to H19 is a distorted doublet with ³J_(HF)=6.9 Hz due tothe spincoupling with the fluorine nuclei in position 18. Carbonresonances corresponding to C17, C18, C19 and C20 also exhibit couplingswith these fluorine nuclei. The C17 and C20 resonances are triplets with²J_(CF)=19 Hz and ³J_(CF)=11 Hz, respectively. The C18 resonance is adoublet of doublets with coupling constants ¹J_(CF)=251 Hz and ³J_(CF)=8Hz. The C19 resonance is a multiplet.

Comparing the magnitudes of integrals for resonances corresponding tothe 1,5-naphtalenedisulfonate counter ion and the mother compound givesthe stoichiometric relation of a single 1,5-naphtalenedisulfonatecounter ion crystallized with two molecules of the mother compound.

¹H and ¹³C NMR chemical shift assignment and proton-proton correlationsare shown in Table 11.

TABLE 11 Through- bond cor- Atom ¹³C shift/ ¹H shift/ppm^(b) andrelation No. Type ppm^(a) multiplicity^(c) J_(HH)/Hz to ¹H^(d)  1 CHF₂117.5^(e) 6.91 (t) 73 (²J_(HF)) nd  1′ 117.5^(e) 6.87 (t) 73 (²J_(HF))nd  2 C 153.5 na na na  2′ 153.3 na na na  3 CH 120.0 7.14 (t)^(n) nd 5,7  3′ 119.6 7.11 (t)^(n) nd 5′, 7′  4 C 136.1 na na na  4′ 135.8 na nana  5 CH 125.0 7.31 (t)^(n) nd 3, 7  5′ 124.9 7.28 (t)^(n) nd 3′, 7′  6C 144.4 na na na  6′ 145.3 na na na  7 CH 117.2 7.16 (t)^(n) nd 3, 5  7′117.1 7.12 (t)^(n) nd 3′, 5′  8 CH  72.9 5.15 (s) na nd  8′  73.6 5.07(s) na nd  9 CO 173.0 na na na  9′ 173.5 na na na 11 CH₂  51.5 a: 4.29(m) b: 4.13 nd 12, 13 (m) 11′  48.6 a: 4.01 (m) b: 3.93 nd 12′, 13′ (m)12 CH₂  21.7 a: 2.46 (m) b: 2.17 nd 11, 13 (m) 12′  22.8 a: 2.61 (m) b:2.03 nd 11′, 13′ (m) 13 CH  62.8 4.70 (dd)  6.0 and 12  9.4 13′  65.85.14 (dd)  5.6 and 12′  9.1 14 CO 172.4 na na na 14′ 173.2 na na na 16CH₂  32.3 4.51 (m) nd nd 16′  32.5 4.51 (m) nd nd 17 C 121.0^(f) na nana 18 CF 162.8^(g) na na na 19 CH 112.7^(i) 7.35 (d)  6.9 (³J_(HF)) nd20 C 127.9^(k) na na na 21 C 160.0 na na na 21′ 159.9 na na na 24 CH₃ 64.8 3.93 (s) na nd 24′  64.8 3.92 (s) na nd 25 C 142.4 na na na 26 CH126.8 8.16 (d)  7.2 27, 28 27 CH 125.9 7.54 (dd)  8.6 and 26, 28  7.2 28CH 131.0 8.97 (d)  8.6 26, 27 29 C 131.1 na na na ^(a)Relative to thesolvent resonance at 49.0 ppm. ^(b)Relative to the solvent resonance at3.30 ppm. ^(c)s = singlet, d = doublet, dd = doublet of doublets, t =triplet, m = multiplet. ^(d)Obtained in the gCOSY experiment. ^(e)Theresonance is a triplet due to coupling with the two fluorine nuclei F1.¹J_(CF) = 263 Hz. ^(f)The resonance is a triplet due to coupling to thetwo fluorine nuclei F18. ²J_(CF) = 19 Hz. ^(g)The resonance is a doubletof doublets due to coupling to the two fluorine nuclei F18. ¹J_(CF) =251 Hz and ³J_(CF) = 8 Hz. ^(i)The resonance is a multiplet due tocoupling to the two fluorine nuclei F18. ^(k)The resonance is a tripletdue to coupling to the two fluorine nuclei F18. ³J_(CF) = 11 Hz. ^(n)The⁴J_(HH) coupling with the meta-protons is not fully resolved. na = notapplicable, nd = not determined

Crystals of Compound B, hemi-1,5-naphtalenedisulfonic acid salt(obtained by way of Method 18-I above, were analyzed by XRPD and theresults are tabulated below (Table 12) and are shown in FIG. 5.

TABLE 12 Intensity d value (Å) (%) Intensity 18.3 99 vs 12.5 22 s 9.9 22s 9.1 67 vs 8.0 18 m 7.5 17 m 6.8 37 s 6.7 59 s 6.1 39 s 6.0 21 s 5.6 66vs 5.5 98 vs 4.94 48 s 4.56 59 s 4.39 35 s 4.27 33 s 4.13 81 vs 4.02 87vs 3.86 88 vs 3.69 69 vs 3.63 100 vs 3.57 49 s 3.48 53 s 3.23 35 s 3.1943 s 3.16 38 s

DSC showed an endotherm with an extrapolated melting onset temperatureof ca 183° C. and TGA showed a 0.3% weight loss between 25–110° C.

Abbreviations Ac = acetyl APCI = atmospheric pressure chemicalionisation (in relation to MS) API = atmospheric pressure ionisation (inrelation to MS) aq. = aqueous Aze(& (S)-Aze) =(S)-azetidine-2-carboxylate (unless otherwise specified) Boc =tert-butyloxycarbonyl br = broad (in relation to NMR) CI = chemicalionisation (in relation to MS) d = day(s) d = doublet (in relation toNMR) DCC = dicyclohexyl carbodiimide dd = doublet of doublets (inrelation to NMR) DIBAL-H = di-isobutylaluminium hydride DIPEA =diisopropylethylamine DMAP = 4-(N,N-dimethyl amino) pyridine DMF =N,N-dimethylformamide DMSO = dimethylsulfoxide DSC = differentialscanning colorimetry DVT = deep vein thrombosis EDC =1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride eq. =equivalents ES = electrospray ESI = electrospray interface Et = ethylether = diethyl ether EtOAc = ethyl acetate EtOH = ethanol Et₂O =diethyl ether HATU = O-(azabenzotriazol-1-yl)-N,N,N′,N′-tetra-methyluronium hexafluorophosphate HBTU =[N,N,N′,N′-tetramethyl-O-(benzotriazol-1- yl)uroniumhexafluorophosphate] HCl = hydrochloric acid, hydrogen chloride gas orhydrochloride salt (depending on context) Hex = hexanes HOAc = aceticacid HPLC = high performance liquid chromatography LC = liquidchromatography m = multiplet (in relation to NMR) Me = methyl MeOH =methanol min. = minute(s) MS = mass spectroscopy MTBE = methyltert-butyl ether NMR = nuclear magnetic resonance OAc = acetate Pab =para-amidinobenzylamino H-Pab = para-amidinobenzylamine Pd/C = palladiumon carbon Ph = phenyl PyBOP =(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate q =quartet (in relation to NMR) QF = tetrabutylammonium fluoride rt/RT =room temperature s = singlet (in relation to NMR) t = triplet (inrelation to NMR) TBTU = [N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate] TEA = triethylamine Teoc =2-(trimethylsilyl)ethoxycarbonyl TEMPO =2,2,6,6-tetramethyl-1-piperidinyloxy free radical TFA = trifluoroaceticacid TGA = thermogravimetric analysis THF = tetrahydrofuran TLC = thinlayer chromatography UV = ultraviolet

Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal,secondary, iso, and tertiary.

The invention is illustrated, but in no way limited, by the followingExamples. Unless otherwise specified, HPMC polymers were obtained fromShin-Etsu (trademark METOLOSE™). For iota-carrageenan the supplier wasFluka for all Examples except Examples 11 and 12 (when CP-Kelco was thesupplier). Specific grades and their USP equivalents are indicated below(once only, on the first occasion that they are disclosed).

General Test Method

Three individual tablets were tested for drug release in 900 ml mediausing a USP dissolution apparatus 2 (paddle+basket¹) at 50 rpm and 37°C. The dissolution media used were 0.1 M hydrochloric acid (pH 1) and0.1 M sodium phosphate buffer (pH 6.8). In-line quantitation wasperformed using the C Technologies fibre optic system with 220 nm as theanalytical wavelength when 0.1 M HCl was used as the dissolution mediaand with 260 nm as the analytical wavelength when phosphate buffer pH6.8 was used as the dissolution media. 350 nm was used as the referencewavelength with both media. For the first two hours of the analysis therelease value was measured every 15 minutes, and then every hour for theremainder of the analysis.

[¹A custom made quadrangular basket of mesh wire, soldered in one of itsupper, narrow sides to the end of a steel rod. The rod is broughtthrough the cover of the dissolution vessel and fixed by means of twoTeflon nuts, 3.2 cm from the centre of the vessel. The lower edge of thebottom of the basket is adjusted to be 1 cm above the paddle. The basketis directed along the flow stream with the tablet under test standing onits edge].

EXAMPLE 1

Direct compression of Compound A with HPMC 10 000 cPs and HPMC 50 cPs,ratio 50:50.

The active substance and excipients material were mixed in a beetingvat. The granulate was lubricated with sodium stearyl fumarate andcompressed into tablets using an excenterpress.

Weight Amount Compound A 50.5 mg 20.0% HPMC 10000 cPs 100.0 mg 39.5%HPMC 50 cPs 100.0 mg 39.5% Sodium stearyl fumarate 2.5 mg  1.0%

Release Data Time (min) % released in buffer pH 1.1 % released in bufferpH 6.8 0 0 0 15 7 1 30 11 2 45 14 3 60 17 4 120 27 7 180 35 10 240 42 13360 55 18 480 65 23 600 74 28 720 81 33 840 86 38 960 93 43 1080 99 471200 105 52

EXAMPLE 2

Granulation and compression of Compound A with HPMC, solubilizing agentand fillers.

The active substance, antioxidant and solubilizer were dissolved inethanol and distributed in the excipients. This mixture was thengranulated with HPC dissolved in ethanol. The granules were then driedin a drying oven. The granulate was lubricated with sodium stearylfumarate and compressed into tablets using an excenterpress.

Weight Amount Compound A 10 mg 4.7% Polyoxyl 40 hydrogenated castor oil10 mg 4.7% Propyl gallate 0.06 mg 0.03%  HPC LF 10 mg 4.7% HPMC 50 cPs70 mg  33% HPMC 10 000 cPs 30 mg  14% Sodium aluminium silicate 47 mg 22% Lactose, anhydrous 28 mg  13% Microcrystalline cellulose 3 mg 1.4%Sodium stearyl fumarate 4 mg 2.0%

Release Data Time (min) % released in buffer pH 1.1 % released in bufferpH 6.8 0 0 0 15 10 8 30 15 10 45 20 13 60 24 16 120 39 26 180 52 36 24064 45 360 83 65 480 98 84 600 108 98 720 111 114 840 113 123 960 114 1291080 117 132 1200 120 132

The results have not been corrected for the significant backgoundabsorption from the tablet matrix, thus release values of 120% and 132%are seen in 0.1 M HCl and 0.1 M sodium phosphate buffer pH 6.8,respectively.

EXAMPLE 3

Granulation and compression of Compound A with HPMC, SDS and fillers.

The active substance and excipients material were mixed in a beetingvat. The granulate was lubricated with sodiumstearylfumarate andcompressed into tablets using an excenterpress.

Weight Amount Compound A 50 mg 23% HPC LF 13 mg  6% HPMC 50 cPs 100 mg47% Mannitol 50 mg 23% Sodium lauryl sulfate 20 mg  9% Sodium dihydrogenphosphate 75 mg 35% dihydrate Sodium stearyl fumarate 3 mg  1%

EXAMPLE 4 An Example of a Formulation Comprising the Esylate Salt ofCompound A, HPMC and SDS

Weight Amount esylate salt of Compound A 50 mg 23% HPC LF 13 mg  6% HPMC50 cPs 100 mg 47% Mannitol 50 mg 23% Sodium lauryl sulfate 20 mg  9%Sodium dihydrogen phosphate dihydrate 75 mg 35% Sodium stearyl fumarate3 mg  1%

The formulation was prepared according to the method of Example 3.

EXAMPLE 5 An Example of a Formulation with Esylate Salt of Compound Aand Xanthan Gum

Weight Amount esylate salt of Compound A 50 mg 19% Xanthan Gum 200 mg80% Sodium stearyl fumarate 2.5 mg  1%

The formulation was prepared according to the method of Example 3.

EXAMPLE 6 Example of a Formulation of the Esylate Salt of Compound Awith HPMC and Iota-Carregeenan

Weight Amount esylate salt of Compound A 500 mg 50% HPMC 10000 cPs 245mg 25% Iota-Carrageenan 245 mg 25% Sodium stearyl fumarate 10 mg  1%

The formulation was prepared according to the method of Example 3.

EXAMPLE 7 Example of a Formulation of the n-propyl Sulphonic Acid Saltof Compound A with HPMC and Iota-Carregeenan

Weight Amount n-propyl sulphonic acid salt of 100 mg 20% Compound A HPMC10000 cPs 150 mg 30% Iota-Carrageenan 250 mg 50% Sodium stearyl fumarate5 mg  1%

The formulation was prepared according to the method of Example 3.

EXAMPLE 8 Example of a Formulation of the Besylate Salt of Compound Awith HPMC and Iota-Carregeenan

Weight Amount besylate salt of Compound A 20 mg 16% HPMC 10000 cPs 50 mg41% Iota-Carrageenan 50 mg 41% Sodium stearyl fumarate 2 mg  2%

The formulation was prepared according to the method of Example 3.

EXAMPLE 9

Direct compression of esylate salt of Compound A with HPMC 10 000 cPsand HPMC 50 cPs, ratio 50:50.

Weight Amount esylate salt of Compound A 50.5 mg 20.0% HPMC 10000 cPs100.0 mg 39.5% HPMC 50 cPs 100.0 mg 39.5% Sodium stearyl fumarate 2.5 mg 1.0%

The active substance and excipients material has been mixed in a beetingvat. The granulate was lubricated with sodium stearyl fumarate andcompressed into tablets using an excenterpress.

Release Data % released in % released in Time (min) buffer pH 1.1 bufferpH 6.8 0 0 0 15 6 2 30 10 4 45 13 5 60 15 6 120 23 9 180 30 12 240 37 15360 48 20 480 57 25 600 65 30 720 72 34 840 78 38 960 83 42 1080 87 461200 90 49

EXAMPLE 10

Direct compression of esylate salt of Compound A with HPMC 10 000 cPsand ratio 50:50.

The active substance and excipients material has been mixed in a beetingvat. The granulate was lubricated with sodiumstearylfumarate andcompressed into tablets using an excenterpress.

Weight Amount esylate salt of Compound A 50.5 mg 20.0% HPMC 10000 cPs100.0 mg 39.5% Iota-carrageenan (Fluka) 100.0 mg 39.5% Sodium stearylfumarate 2.5 mg  1.0%

Release Data % released in % released in Time (min) buffer pH 1.1 bufferpH 6.8 0 0 0 15 3 2 30 5 3 45 6 5 60 8 6 120 15 13 180 22 21 240 29 30360 43 51 480 57 72 600 70 91 720 81 104 840 88 106 960 94 106 1080 96106 1200 96 106

EXAMPLE 11

The besylate salt of Compound A and excipient materials were granulatedin a high shear granulator. The granulate was dried and lubricated withsodiumstearylfumarate and compressed into tablets using anexcenterpress.

Weight Amount Besylate salt of Compound A 230 mg 57%  HPMC 10000 cPs 17mg 4% HPMC 50 cPs 12 mg 3% Iota-Carrageenan 141 mg 35%  Sodium stearylfumarate 4 mg 1%

Release Data % released in % released in Time (hours) buffer pH 1.1buffer pH 6.8 0 0 0 0.5 6 5 1 10 12 1.5 14 20 2 19 28 3 27 45 4 35 61 542 76 6 — 87 7 — 95 8 — 98 9 — 98 10 — 98 12 — 98

EXAMPLE 12

The besylate salt of Compound A and excipient materials were granulatedin a high shear granulator. The granulate was dried and lubricated withsodiumstearylfumarate and compressed into tablets using anexcenterpress.

Weight Amount Besylate salt of Compound A 230 mg 45% HPMC 10000 cPs 14mg  3% HPMC 50 cPs 163 mg 32% Iota-Carrageenan 94 mg 19% Sodium stearylfumarate 5 mg  1%

Release Data % released in % released in Time (hours) buffer pH 1.1buffer pH 6.8 0 0 0 0.5 5 4 1 10 8 1.5 16 12 2 21 16 3 32 24 4 43 32 554 39 6 — 46 7 — 53 8 — 60 9 — 66 10 — 71 12 — 81 14 — 88 16 — 94 18 —98 20 — 100

EXAMPLE 13

The besylate salt of Compound A and excipient materials were directlycompressed.

Weight Amount Besylate salt of Compound A 263 mg 65% HPMC 50 cPs 137 mg34% Sodium stearyl fumarate 4 mg  1%

Release Data % released in % released in Time (min) buffer pH 1.1 bufferpH 6.8 0 0 0 15 3 4 30 8 7 45 13 10 60 18 14 75 23 17 90 27 20 105 32 23120 36 26 150 44 32 180 51 37 210 59 42 240 66 47 270 72 52 300 78 56330 — 60 360 — 64 390 — 67 420 — 70 450 — 74 480 — 76 510 — 79 540 — 82570 — 84 600 — 86

EXAMPLE 14

Examples of formulations comprising the besylate salt of Compound A.Examples 14-A and 14-B are prepared according to the general method ofExample 3 or 11.

EXAMPLE 14-A Weight Amount Besylate salt of Compound A 50 mg 23% HPC LF13 mg  6% HPMC 50 cPs 100 mg 47% Mannitol 50 mg 23% Sodium laurylsulfate 20 mg  9% Sodium dihydrogen 75 mg 35% phosphate dihydrate Sodiumstearyl fumarate 3 mg  1%

EXAMPLE 14-B Weight Amount Besylate salt of Compound A 150 mg 35%PolyethylenoxideWSR N-60K 200 mg 47% Microcrystalline cellulose 60 14%Polyvinyl pyrrolidone K30 10 mg  2% Sodium stearyl fumarate 3 mg  1%

EXAMPLE 14-C Weight Amount Besylate salt of Compound A 132 mg 30%PolyethylenoxideWSR N-60K 200 mg 69% Sodium stearyl fumarate 4 mg  1%

The formulation was prepared according to the method of Example 3.

Release Data for Example 14-C % released in Time (hours) buffer pH 6.8 00 0.5 3 1 5 1.5 7 2 9 2.5 11 3 13 4 18 5 23 6 28 7 33 8 38 9 44 10 49 1154 12 60 13 65 14 70 15 74 16 78 17 82 18 86 19 89 20 92 21 95 22 97 2399 24 100

EXAMPLE 15

Examples of formulations comprising the hemi-naphthalene 1,5-disulphonicacid salt of Compound B. All formulations are prepared according to thegeneral method of Example 11.

EXAMPLE 15-A Weight Amount Hemi-naphthalene 1,5- 20 mg 16% disulphonicacid salt of Compound B HPMC 10000 cPs 50 mg 41% Iota-Carrageenan 50 mg41% Sodium stearyl fumarate 2 mg  2%

EXAMPLE 15-B Weight Amount Hemi-naphthalene 1,5- 200 mg 44% disulphonicacid salt of Compound B PolyethylenoxideWSR N-60K 250 mg 55% Sodiumstearyl fumarate 4 mg  1%

EXAMPLE 15-C Weight Amount Hemi-naphthalene 1,5- 200 mg 40% disulphonicacid salt of Compound B HPMC 10000 cPs 120 mg 24% HPMC 50 cPs 180 mg 36%Sodium stearyl fumarate 5 mg  1%

Further Examples may be prepared as above in which HPMC is replaced by,or mixed with, PEO. In case of a mixture, the proportion of PEO:HPMC mayrange from 90:10 to 10:90%. A particular mixture is PEO:HPMC 80%:20%, or75%:25%.

EXAMPLE 16

Granulation and compression of Compound A with HPMC, SDS and fillers.The active substance and excipients material were mixed in a beetingvat. The granulate was lubricated with sodiumstearylfumarate andcompressed into tablets using an excenterpress.

Weight Amount Compound A 48 mg 18% HPC LF 13 mg  5% HPMC 50 cPs 60 mg22% Mannitol 50 mg 19% Sodium lauryl sulfate 20 mg  7% Sodium dihydrogen75 mg 28% phosphate dihydrate Sodium stearyl fumarate 3 mg  1%

Release Data % released in % released in Time (min) buffer pH 1.1 bufferpH 6.8 0 0 0 15 9 9 30 19 24 45 28 43 60 37 63 120 70 109 180 92 114 240103 115 360 106 116 480 106 116 600 102 116 720 100 116 840 100 116 96099 116 1080 99 116 1200 99 116

EXAMPLE 17

Direct compression of Compound A with Xanthan Gum and iota-Carrageenan,ratio 50:50.

The active substance and excipients material were mixed in a beetingvat. The granulate was lubricated with sodiumstearylfumarate andcompressed into tablets using an excenterpress.

Weight Amount Compound A 50.5 mg 20.0% ι-Carrageenan 100.0 mg 39.5%Xanthan Gum 100.0 mg 39.5% Sodium stearyl fumarate 2.5 mg  1.0%

Release data % released in % released in Time (min) buffer pH 1.1 bufferpH 6.8 0 0 0 15 2 1 30 4 1 45 5 2 60 7 3 120 12 6 180 16 11 240 21 17360 30 31 480 39 45 600 48 60 720 56 75 840 63 88 960 70 97 1080 75 1001200 79 100

Any Example above which uses the free base or a salt other than thebesylate salt of Compound A may be repeated using the besylate salt ofCompound A.

Particular aspects of the invention are provided as follows:

-   1. A modified release pharmaceutical composition comprising, as    active ingredient, a compound of formula (I):

-   -   R¹ represents C₁₋₂ alkyl substituted by one or more fluoro        substituents;    -   R² represents hydrogen, hydroxy, methoxy or ethoxy; and    -   n represents 0, 1 or 2;    -   or a pharmaceutically acceptable salt thereof; and a        pharmaceutically acceptable diluent or carrier; provided that        the formulation may only contain iota-carrageenan and a neutral        gelling polymer when the compound of formula (I) is in the form        of a salt.

-   2. A composition as described in aspect 1 wherein the active    ingredient is a salt of:    -   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) (Compound A);    -   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe)        (Compound B); or,    -   Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) (Compound C).

-   3 A composition as described in aspect 1 or 2 wherein the active    ingredient is a crystalline salt of:    -   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) (Compound A);    -   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe)        (Compound B);    -   or, Ph(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) (Compound        C).

-   4. A composition as described in aspect 1, 2 or 3 wherein the    composition comprises a gelling matrix.

-   5. A composition as described in aspect 4 wherein the matrix    comprises HPMC.

-   6. A composition as described in aspect 4 or 5 wherein the matrix    comprises iota-carrageenan.

-   7. A composition as described in aspect 4 wherein the matrix    comprises SDS.

-   8. A composition as described in aspect 2 and 5 or 2 and 6 wherein    the matrix additionally comprises xanthan gum.

-   9. The use of a formulation as described in aspect 1 as a    medicament.

-   10. The use of a formulation as described in aspect 1 in the    manufacture of a medicament for the treatment of a cardiovascular    disorder.

-   11. A method of treating a cardiovascular disorder in a patient    suffering from, or at risk of, said disorder, which comprises    administering to the patient a therapeutically effective amount of a    pharmaceutical formulation as described in aspect 1.

-   12. A process for making an immediate release formulation as    described in aspect 1.

Also provided is a composition obtainable by any of the Methods and/orExamples described herein.

1. A modified release pharmaceutical composition comprising apharmaceutically acceptable salt of a compound of formula (I) as activeingredient

wherein: R¹ is —CHF₂ or —CH₂CH₂F; R² is hydrogen, hydroxy, methoxy orethoxy; and n is 0 or 2; and at least one gelling polymer selected fromhydroxymethylcellulose (HMC), hydroxyethylcellulose (HEC),hydroxypropylcellulose (HPC), methylcellulose (MC), ethylcellulose (EC),carboxyethylcellulose (CEC), ethylhydroxyethylcellulose (EHEC),carboxymethylhydroxyethylcellulose (CMHEC), hydroxypropylmethylcellulose(HPMC), hydroxypropylethylcellulose (HPEC) and sodiumcarboxymethylcellulose (Na CMC).
 2. The composition according to claim 1wherein the gelling polymer comprises HPC and HPMC.
 3. The compositionaccording to claim 2 wherein the HPMC is present in about 30 to about 80percent w/w.
 4. The composition according to claim 1 wherein the gellingpolymer comprises HPC and EC.
 5. The composition according to claim 4wherein the HPC is present in about 10 to about 95 percent w/w.
 6. Thecomposition according to claim 4 wherein the HPC is present in about 30to about 80 percent w/w.
 7. The composition according to any one ofclaims 1, 2 and 4 further comprising at least one pharmaceuticallyacceptable diluent and/or lubricant.
 8. The composition according toclaim 7 wherein the pharmaceutically acceptable diluent and/or lubricantis selected from calcium phosphate, lactose, microcrystalline cellulose,mannitol, sorbitol, titanium dioxide, aluminum silicate, magnesiumstearate, sodium stearyl fumarate and mixtures thereof.
 9. Thecomposition according to claim 1 wherein the active ingredient is apharmaceutically acceptable salt ofPh(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe);Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe); orPh(3-Cl)(5-OCH₂CH₂F)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe).
 10. The compositionaccording to claim 9 wherein the pharmaceutically acceptable salt iscrystalline.
 11. The composition according to any one of claims 1, 9 and10 wherein the pharmaceutically acceptable salt is an acid additionsalt, wherein the acid of the acid addition salt is selected from thegroup consisting of ethanesulfonic acid, n-propanesulfonic acid,benzenesulfonic acid, 1,5-naphthalenedisulfonic acid andn-butanesulfonic acid.
 12. The composition according to claim 11 whereinthe active ingredient is benzenesulfonic acid salt ofPh(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(OMe) characterised by anX-ray powder diffraction pattern characterised by peaks with d-values at5.9, 4.73, 4.09 and 4.08Å.
 13. The composition according to claim 11wherein the active ingredient is hemi-1,5-naphthalenedisulfonic acidsalt of Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe)characterised by an X-ray powder diffraction pattern characterised bypeaks with d-values at 18.3, 9.1, 5.6, 5.5, 4.13, 4.02, 3.86, 3.69 and3.63Å.
 14. The composition according to claim 1 further comprisingiota-carrageenan.
 15. The composition according to claim 1 furthercomprising sodium dodecyl sulphate (SDS).
 16. The composition accordingto claim 1 wherein the composition is formulated as a gel matrix.
 17. Amethod of treating a cardiovascular disorder in a patient suffering fromsaid disorder comprising administrating to the patient a therapeuticallyeffective amount of a composition according to claim 1.